JP2021505131A - Immunotherapy with enhanced iPSC-derived effector cells - Google Patents

Abstract

Methods and compositions for obtaining functionally enhanced inducible effector cells obtained from directed differentiation of genome-engineered iPSCs are provided. The induced cells provided herein are equipped with stable and functional genome editing that results in improved or enhanced therapeutic effects. Also provided are therapeutic compositions and their use that include only functionally enhanced inducible effector cells or that include combination therapy with antibodies or checkpoint inhibitors. [Selection diagram] Fig. 1

Description

Related Applications This application is a US provisional patent application No. 62 / 596,659 filed on December 8, 2017, and a US provisional patent application No. 62 / 657,626 filed on April 13, 2018. Claim the priority of issues and their disclosures are incorporated herein by reference in their entirety.

References to Electronically Submitted Sequence Listings This application is, by reference, submitted with this application, prepared on 30 November 2018, in size of 36,336 bytes, 13601-195-228_SEQ_LISTING. .. Incorporates a computer-readable format (CRF) of ASCII text sequence listings, entitled txt.

The present disclosure relates broadly to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically appropriate properties in vivo. Cell products developed under the present disclosure address significant limitations of patient-derived cell therapies.

The field of adoptive cell therapy is currently focused on using cells from patients and donors, so achieving consistent production of cancer immunotherapy and all that may benefit. Providing treatment to patients has become particularly difficult. There is also a need to improve the efficacy and persistence of adopted transplanted lymphocytes to promote good outcomes for patients. Lymphocytes such as T cells and natural killer (NK) cells are powerful antitumor effectors that play important roles in innate and adaptive immunity. However, the use of these immune cells for adoptive cell therapy remains difficult and there are unmet demands for improvement. Therefore, there remains an important opportunity to maximize the potential of T and NK cells, or other lymphocytes, in adoptive immunotherapy.

Response rate, cell depletion, loss of transfused cells (survival and / or persistence), tumor escape due to loss of target or lineage, accuracy of tumor targeting, non-target toxicity, extratumor effect, solid tumor There is a need for functionally improved effector cells that address a variety of issues such as tumor microenvironment and associated immunosuppression, recruitment, transport, and infiltration.

An object of the present invention is to provide a method and composition for producing induced non-pluripotent cells differentiated from a single cell-derived iPSC (induced pluripotent stem cell) clone strain, and the iPSC strain is a genome thereof. Includes one or several genetic modifications. The aforementioned one or several genetic modifications include DNA insertions, deletions, and substitutions, which are retained in the subsequent indels after differentiation, proliferation, passage and / or transplantation. , Continue to work.

The iPSC-derived non-pluripotent cells of the present application include CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, etc. Includes, but is not limited to, NKT cells, NK cells, and B cells. The iPSC-derived non-pluripotent cells of the present application contain one or several genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modification. In an engineered clonal iPSC differentiation strategy to obtain genetically engineered induced cells, the potential for iPSC development in the indicated differentiation is not adversely affected by the iPSC engineered modality, and the engineered modality. It is also necessary for the induced cells to function as intended. In addition, this strategy is the current barrier to manipulating primary lymphocytes such as T cells or NK cells derived from peripheral blood, i.e. such cells often lack reproducibility and uniformity and are high cells. It results in cells exhibiting inadequate cell persistence with death and low cell proliferation, overcoming the barriers caused by the difficulty of manipulating such cells. In addition, this strategy avoids the generation of heterogeneous effector cell populations that would otherwise be obtained using a primary cell source that is initially heterogeneous.

Some aspects of the invention use methods that include (I), (II), or (III), respectively, following and prior to the reprogramming process, reflecting genomic manipulation strategies. Providing the resulting genome-engineered iPSC:

(I): Gene manipulation of iPSCs and / or both of (i) and (ii) in any order: (i) One or more constructs were introduced into the iPSC and targeted at selected sites. Enables integration; (ii) (a) Introduces one or more double-stranded breaks into the iPSC at the selected site using one or more endonucleases capable of recognizing the selected site; (B) The iPSCs of steps (I), (ii) and (a) are cultured to allow endogenous DNA repair to generate targeted indels at selected sites; thereby partial or complete. Obtain a genome-engineered iPSC capable of differentiating into cells that have differentiated into.

(II): Reprogrammed non-pluripotent cells are genetically engineered to obtain genome-engineered iPSCs: (i) Non-pluripotent cells are one or more reprogramming factors, and optionally Contact with a small molecule composition comprising a TGFβ receptor / ALK inhibitor, MEK inhibitor, GSK3 inhibitor and / or ROCK inhibitor to initiate reprogramming of non-pluripotent cells; (ii) step (II). Introduce one or both of (a) and (b) into reprogrammed non-pluripotent cells in contact with and in any order: (a) Targeted integration at selected sites One or more constructs that enable; (b) After making one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, the step (II) The cells of (ii) and (b) are cultured to allow endogenous DNA repair to produce targeted indels at selected sites; therefore, the acquired genome-engineered iPSCs The aforementioned genome-engineered iPSCs, which include at least one functionally targeted genome edit, can differentiate into partially or fully differentiated cells.

(III): Genetically engineer non-pluripotent cells for reprogramming to obtain genome-engineered iPSCs containing (i) and (ii): (i) to non-pluripotent cells (a) And (b) one or both are introduced in any order: (a) one or more constructs that allow targeted integration at the selected site; (b) recognition of the selected site One or more double-strand breaks are made at selected sites using at least one possible endonuclease, where cells from steps (III), (i) and (b) are cultured to repair endogenous DNA. Allows the generation of targeted indels at selected sites; (ii) cells in steps (III) and (i) with one or more reprogramming factors, and optionally TGFβ receptor / Genome-engineered iPSCs comprising contact with a small molecule composition comprising an ALK inhibitor, MEK inhibitor, GSK3 inhibitor and / or ROCK inhibitor and at least one functionally targeted genome edit at a selected site. The above-mentioned genome-engineered iPSC can be differentiated into partially differentiated cells or fully differentiated cells.

In one embodiment of the method described above, at least one targeted genome edit at one or more selected sites is a safety switch protein, targeted modality, receptor, signaling molecule, transcription factor, pharmaceutical activity. Proteins and peptides encode proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or viability of drug target candidates or genomically engineered iPSCs or their inducible cells1 Includes insertion of one or more exogenous polynucleotides. In some embodiments, the exogenous polynucleotide for insertion is (1) CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, transient, tissue-specific, or cell type. One or more extrinsic promoters, including specific promoters; or (2) meet the criteria of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1, or Genome Safe Harbor. It is operably linked to one or more endogenous promoters contained at selected sites containing the locus of. In some embodiments, the genome-engineered iPSC produced using the method described above is one or more different encoding a protein comprising caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B-cell CD20. If the genome-engineered iPSC comprises two or more suicide genes, the suicide genes are AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta2 microglobulin, GAPDH. , TCR, or RUNX1 in various safeharbor loci. In one embodiment, the exogenous polynucleotide comprises a partial or complete peptide of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. Code. In some embodiments, partial or complete receptors for IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors encoded by exogenous polynucleotides. Peptides are in the form of fusion proteins.

In some other embodiments, genomically engineered iPSCs generated using the methods provided herein are targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immunity. Indel in one or more endogenous genes associated with proteins that suppress response regulation and regulation, or engraftment, transport, homing, viability, self-renewal, persistence, and / or viability of iPSCs or their induced cells. Including. In some embodiments, the endogenous gene for disruption is of any of the genes in the B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and chromosome 6p21 regions. Includes at least one of.

In yet some other embodiments, the genome-engineered iPSC produced using the methods provided herein is at the AAVS1 locus, a caspase encoding an exogenous polynucleotide, and at the H11 locus. Includes thymidine kinase, which encodes an exogenous polynucleotide.

In yet some other embodiments, approaches (I), (II) and / or (III) make the genome-engineered iPSC a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. It further includes maintaining the pluripotency of the genome-engineered iPSC in contact with objects. In one embodiment, the acquired genome-engineered iPSC containing at least one targeted genome editing is functional, capable of differentiating, and differentiated into non-pluripotent cells containing the same functional genome editing. can do.

The present invention also provides:

One aspect of the present application is a cell or population thereof, which is obtained from differentiating any of the induced pluripotent cells (iPSCs), cloned iPSCs, or iPS cell line cells, or the iPSCs described above. Induced cells to be; any of the above cells are high-affinity non-cleavable CD16 (hnCD16) or variants thereof; and (2) chimeric antigen receptors (CAR), and exogenous cytokines or cell surface-expressed exogenous cytokines thereof. Provides a cell or population thereof comprising one or both of a partial or complete peptide of a receptor. In some embodiments of the inducible cells obtained from iPSC differentiation, the inducible cells are CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cells. Hematopoietic cells, including, but not limited to, progenitor cells, T cells, NKT cells, NK cells, and B cells; these hematopoietic cells (ie, induced CD34 cells, induced hematopoietic endothelial cells, induced hematopoietic stem cells and progenitor cells, Induced hematopoietic pluripotent progenitor cells, inducible T cell progenitor cells, inducible NK cell progenitor cells, inducible T cells, inducible NKT cells, inducible NK cells, or inducible B cells) are either peripheral blood, umbilical cord blood, or any other. Contains longer telomeres compared to its native corresponding cells obtained from donor tissue.

In some embodiments of the iPSC and its inducible cells described above, the cell comprises hnCD16 or a variant thereof, CAR, and optionally a partial or complete peptide of an exogenous cytokine or receptor thereof expressed on the cell surface. Further comprises one or more of the following genomic edits: (i) B2M null or low; (ii) CIITA null or low; (iii) introduced expression of HLA-G or non-cleavage HLA-G; (Iv) At least one of the genotypes listed in Table 1; (v) TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any of the chromosome 6p21 regions. in at least one deletion or decreased expression of the gene; and (vi) HLA-E, 41BBL , CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, a 2A R, CAR Introduced or increased expression in at least one of the TCR, Fc receptor, engager, and surface-triggered receptor for binding bispecific or multispecific or universal engagers.

In some embodiments of the aforementioned iPSCs and their inducible cells, including at least hnCD16 or variants and CARs, and optionally additional genomic editing described above and throughout the application, the cells are (i) It may include one or more exogenous polynucleotides integrated into one safe harbor locus, or (ii) more than two exogenous polynucleotides integrated into different safe harbor loci. In some embodiments, the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. In one particular embodiment, the safe harbor locus TCR is the constant region of TCR alpha.

In some embodiments of cells or populations thereof, high affinity non-cleavable CD16 (hnCD16), CAR, with or without partial or complete peptides of exogenous cytokines or receptors thereof expressed on the cell surface. And optionally, the cell comprising one or more of the additional genome edits described above is an inducible NK or inducible T cell, which is at least one of the following characteristics: (I) Persistence and / or viability when compared to its native corresponding NK or T cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, including, but not limited to: Improvement; (ii) increased resistance to spontaneous immune cells; (iii) increased cytotoxicity; (iv) improved tumor penetration; (v) enhanced or acquired ADCC; (vi) tumor bystander immune cells Enhanced ability to migrate to and / or activate or recruit sites; (vii) enhanced ability to reduce tumor immunosuppression; (viii) enhanced ability to rescue tumor antigen escape.

In one embodiment of the cell or population thereof, the cell containing hnCD16 or a variant thereof comprises at least one of the following: (a) derived from F176V and S197P in the external domain domain of CD16; (b) CD64. Complete or partial external domain; (c) non-natural (or non-CD16) transmembrane domain; (d) non-natural (or non-CD16) intracellular domain; (e) non-natural (or non-CD16) Signaling domains; (f) Non-naturally occurring stimulating domains; (g) Transmembrane, signaling, and stimulating domains that are not derived from CD16 but from the same or different polypeptides. In some embodiments, the non-natural transmembrane domains are CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM. -1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) poly Derived from the peptide. In some embodiments, the non-natural stimulation domain is CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. Derived from. In some other embodiments, the non-natural signaling domains are CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. Derived from a polypeptide. In some specific embodiments of the hnCD16 variant, the non-natural transmembrane domain is derived from NKG2D, the non-natural stimulus domain is from 2B4, and the non-natural signaling domain is from CD3ζ.

In one embodiment of the cell or population thereof, the cell comprises hnCD16 or a variant thereof and CAR, which can be one or more of the following: (i) T cell specific or NK cell specific; ii) Bispecific antigen-binding CAR; (iii) switchable CAR; (iv) dimerized CAR ;; (v) split CAR; (vi) multi-chain CAR; (vii) inducible CAR; (Viii) Co-expression with another CAR; (ix) with a partial or complete peptide of an exogenous cytokine or receptor thereof expressed on the cell surface, optionally in a separate construct or in a bicistronic construct. Co-expressed; (xi) co-expressed with a checkpoint inhibitor in a separate construct or bicistronic construct as needed; (xi) CD19 or BCMA specific; and / Or (xiii) ADGRE2, carbonate dehydration enzyme IX (CALX), CCR1, CCR4, cancer fetal antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41 , CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens of cytomegalovirus (CMV) -infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 ( EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folic acid binding protein (FBP), fetal acetylcholine receptor (AChR) , Folic acid receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epithelial growth factor receptor 2 (HER-2), human telomerase reverse transcription enzyme (hTERT), ICAM-1, integrin B7, interleukin -13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1- CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, Mutin 1 (Muc-1), Mutin 16 (Muc-16), Mesocerin (MSLN), NKCSI, NKG 2D ligand, c-Met, carcinoembryonic antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-related glycoprotein 72 ( Specific to any one of TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigen.

In some embodiments where the checkpoint inhibitor is co-expressed with CAR, the checkpoint inhibitor is PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4- 1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR- 1. Antagonist to one or more checkpoint molecules including MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. .. Checkpoint inhibitors co-expressed with CAR are antibodies specific for any of the above checkpoint molecules, or humanized or Fc-modified variants or fragments thereof and their functional equivalents and biosimilars. Can be. In some embodiments, the CAR of any one of (i)-(ix) can be inserted into the TRAC locus. In some embodiments, the CAR of any one of (i)-(ix) inserted at the TRAC locus can be driven by the endogenous promoter of TCR. In some embodiments, insertion of CAR at any one of (i)-(ix) at the TRAC locus results in TCR knockout.

In one embodiment of a cell or population thereof, a cell comprising hnCD16 or a variant thereof and CAR further comprises a partial or complete peptide of an exogenous cytokine or receptor thereof expressed on the cell surface, the exogenous cytokine or receptor thereof. Can comprise at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (i) use self-cleaving peptides. Co-expression of IL15 and IL15Rα; (ii) fusion protein of IL15 and IL15Rα; (iii) fusion protein of IL15 / IL15Rα in which the intracellular domain of IL15Rα was cleaved; (iv) fusion protein of membrane-bound cytokine domain of IL15 and IL15Rα (V) IL15 and IL15Rβ fusion protein; (vi) IL15 and common receptor γC fusion protein, where the common receptor γC is native or modified; (vii) at least one of the IL15Rβ homodimers. Can be included, wherein any one of (i)-(vii) can be co-expressed with CAR in a separate construct or in a bicystonic construct. In some embodiments, partial or complete peptides of cell surface exogenous cytokines or receptors are transiently expressed in the cells provided herein.

In one embodiment of the cell or population thereof, the cell containing hnCD16 or a variant thereof and CAR is an inducible NK cell or an inducible T cell, which can mobilize and / or migrate T cells to the tumor site. Yes, where induced NK cells or induced T cells can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. In some embodiments, the checkpoint inhibitors are PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA. , CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT -2, an antagonist against one or more checkpoint molecules including Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some other embodiments, checkpoint inhibitors are (a) atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monarizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof. Includes one or more of them, or at least one of (b) atezolizumab, nivolumab, and pembrolizumab.

Another aspect of the application provides a composition comprising any cell or population thereof described above and throughout the application. In some embodiments, the iPSC or iPSC-derived cells (inducible cells) may comprise any one of the genotypes listed in Table 1 of the present application. In some embodiments, the iPSC or derived cells from it comprises hnCD16 and CAR. In some embodiments, the iPSC or derived cells from it are a partial or complete peptide of the exogenous cytokines or receptors thereof for hnCD16, CAR, and cell surface expression described above and throughout the application. Including. In some embodiments of cells containing hnCD16 and CAR, CAR is specific for CD19. In other embodiments of cells containing hnCD16 and CAR, CAR is specific for CD269 (BCMA). In yet some other embodiments, the CAR is ADGRE2, carbonate dehydration enzyme IX (CALX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30. , CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, Antigens of cytomegalovirus (CMV) -infected cells (eg, cell surface antigens) , Epithelial glycoprotein 2 (EGP2), Epithelial glycoprotein-40 (EGP-40), Epithelial cell adhesion molecule (EpCAM), EGFRvIII, Receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB Folic acid binding protein (FBP), fetal acetylcholine receptor (AChR), folic acid receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epithelial growth factor receptor 2 (HER-2), human telomerase reversal Photoenzyme (hTERT), ICAM-1, Integrin B7, Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9) ), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, Mutin 1 (Muc-1), Mutin 16 (Muc-16) , Mesocerin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testile antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA) ), Tumor-related glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various known in the art. It is specific for any one of the pathogenic antigens.

Therefore, a further aspect of the application provides a composition for therapeutic use that comprises one or more therapeutic agents in addition to any of the inducible cells provided herein. In some embodiments of compositions for therapeutic use, the therapeutic agent is a peptide, cytokine, checkpoint inhibitor, mitogen, growth factor, small RNA, dsRNA (double-stranded RNA), mononuclear blood cells, Includes feeder cells, feeder cell components or replacement factors thereof, vectors, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulators (IMiD) containing one or more polypeptides of interest. In some embodiments of compositions for therapeutic use, the checkpoint inhibitors used with the cells provided are PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-. 4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, One or more checks including EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR Includes antagonists to point molecules. In some embodiments of the composition for therapeutic use, the checkpoint inhibitors used with the cells provided are atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monarizumab, nivolumab, Includes pembrolizumab and one or more of its derivatives or functional equivalents. In some other embodiments of the composition for therapeutic use, the checkpoint inhibitor used with the cells provided comprises atezolizumab, nivolumab, and at least one of pembrolizumab.

In some embodiments of the composition for therapeutic use, the antibodies used with the cells provided are anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and /. Alternatively, it comprises any one of anti-CD38 antibodies. In some embodiments of the composition for therapeutic use, the antibodies used with the cells provided are rituximab, beltuzumab, ofatumumab, ubrituximab, okaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, sertuximab, dinutuximab, Includes avelumab, daratumumab, isatumumab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, as well as one or more of their functional equivalents and biosimilars. In yet some other embodiments of the composition for therapeutic use, the antibody used with the cells provided comprises daratumumab.

The application also provides the therapeutic use of the cells or therapeutic compositions described herein by introducing the composition into a subject suitable for adoptive cell therapy. In some embodiments, the subject suitable for and in need of adoptive cell therapy has an autoimmune disorder, hematological malignancies, solid tumors; cancer, or viral infection.

A further aspect of the application is a method of producing the inducible cells described herein, comprising differentiating an iPSC comprising hnCD16 and CAR, and optionally one or more of the following: Provided: (i) B2M null or low; (ii) CIITA null or low; (iii) introduced expression of HLA-G or non-cleavage HLA-G; (iv) exogenous cytokines of cell surface expression or their thereof. Partial or complete peptide of the receptor; (v) at least one of the genotypes listed in Table 1; (vi) TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, Deletion or underexpression in at least one of the RFX5, RFXAP, and any genes in the 6p21 region of the chromosome; and (vii) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137. , _CD80, PDLl, a 2A R, definitive CAR, TCR, Fc receptors, engagers, and to at least one of the bispecific or multispecific or universal engagers and binding for surface triggering receptor Introduced or increased expression.

In some embodiments of the method of manufacture, the method is to insert an hnCD16 or variant thereof, or CAR, optionally to knock out B2M and CIITA, or HLA-G or non-cleavable HLA. It further comprises genomic manipulation of cloned iPSCs to introduce -G and / or to introduce partial or complete peptides of exogenous cytokines or receptors thereof expressed on the cell surface. In some embodiments, the two modalities are in separate constructs or in bicistronic constructs for cells containing both CAR and exogenous cytokines of cell surface expression or partial or complete peptides of their receptors. , Co-expressed. In some embodiments of the method of manufacture, genomic manipulation of iPSCs comprises targeted editing. In some embodiments, the targeted edit comprises delete, insert, or indel. In some embodiments, targeted editing is performed by CRISPR, ZFN, TALEN, homing nucleases, homologous recombination, or any other functional variation of these methods.

The application further provides editing of CRISPR-mediated cloned iPSCs, thereby containing at least one of hnCD16 or its variants and CAR, or the genotypes listed in Table 1. To generate. All genotypes listed in Table 1 include insertions of hnCD16 and CAR.

A further aspect of the application is a method of preventing or reducing tumor antigen escape and / or tumor recurrence, in which hnCD16 or a variant thereof, CAR, and optionally, a partial of a cytokine or receptor thereof expressed on the cell surface. Or administer to a subject under treatment either an effector cell containing the complete peptide, and either an antigen-specific monoclonal antibody, or a humanized or Fc-modified variant or fragment, a functional equivalent and their biosimilars. Antigens targeted by antibodies, including, provide a method different from tumor antigens recognized by CAR. In some embodiments, the effector cell comprises at least one of the genotypes listed in Table 1.

The various objectives and advantages of the compositions and methods provided herein will become apparent from the following description, along with accompanying drawings showing specific embodiments of the invention by way of example and by way of example.

Illustration of several construct designs for cell surface expressed cytokines or their receptors in iPSC-derived cells. IL15 is used as an exemplary example in which it can be replaced with other desirable cytokines. A graph of flow cytometry of mature iPSC-derived NK cells, the stages of hnCD16 expression, B2M knockout (loss of HLA-A2 expression), HLA-G expression, and IL-15 / IL-15ra (LNGFR) construct expression. Indicates a target operation. A graphical representation of telomere length determined by flow cytometry, iPSC-derived maturation-induced NK cells maintain longer telomeres compared to adult peripheral blood NK cells. We show that B2M knockout eliminates in vitro recognition of engineered induced NK cells by allogeneic CD8 + T cells. It is shown that expression of HLA-G rescues B2M ^{− / −} iPSC from killing by NK cells. A: Loss of HLA-I results in increased cytotoxicity to induced iPSCs when incubated with allogeneic PBMCs. B: Expression of HLA-G on B2M ^{− / −} iPSC partially reversed the killing of allogeneic manipulated iPSCs. IPSC loss was measured over time using the Incute Zoom ™ imaging system, and the data were normalized to the number of iPSCs in effector cell-free wells, with 0-100% time under each condition. Set to. It is shown that a single dose of hnCD16 / B2M ^{− / −} HLA-GINK induced tumor regression in an in vivo xenograft model of ovarian cancer. A: IVIS image of each mouse 32 days after injection. B: Time course of tumor progression by IVIS imaging. It is shown that the IL15 / IL15Ra construct promotes iNK cell differentiation and viability in vitro, independent of the addition of soluble exogenous IL15. A: iNK cells of each of the indicated genotypes differentiated with or without the addition of soluble IL15. B: iNK cells were thoroughly washed and returned to culture for 7 days in soluble IL15 at concentrations ranging from 10 ng / ml to 0 ng / ml to observe soluble IL15-independent cell growth. It is shown that the expression of IL15 / IL15Ra construct enhances the persistence of iNK in vivo in the absence of soluble IL15. iNK cells were adopted into A: immunodeficient NOG mice and B: transgenic NOG mice with human IL15. FIG. 3 is a graph representation of the phenotype of CAR-expressing iPSC-derived NK cells using flow cytometric analysis of surface markers. It is a graph display of the antitumor activity of CAR4 expression-inducing NK cells co-cultured with europium-loaded meso-high target cells in various effector-to-target (E: T) ratios. A: K562meso-high target cell; B: A1847 meso-high target cell. In a xenograft NSG mouse model inoculated with a single dose of luciferase-expressing A1847 mesohigh cells and 1.5E7 NK cells of different genotypes 4 days after A1847 inoculation, the tumor volume determined by weekly bioluminescence imaging is shown. A: Quantified tumor volume determined using bioluminescence, and B: A single dose of A1847 mesohigh cells expressing luciferase and 1.5E7 NK cells of different genotypes 4 days after A1847 inoculation. The Kaplan-Meier curve representing the survival percentage of the inoculated NSG mouse group is shown. N = 5 in all groups. Enhancement of CAR-iNK in vivo by measuring the percentage of induced CAR-NK cells derived from cells collected from (A) peripheral blood, (B) spleen, and (C) peritoneum as assessed by flow cytometry. Shows sustained persistence. Each dot represents one recipient mouse. The median ± SEM is shown and P <0.05. FIG. 5 is a graph representation of the creation of CD16 expression-inducing T cells from a clonal population of hnCD16-iPSCs. A: hnCD16-iPSC flow analysis. B: hnCD16-iT flow analysis. It shows the elimination of target cells via ADCC by hnCD16-iT cells. It is shown that expression of both CAR and hnCD16 does not disrupt the hematopoietic differentiation of CAR-hnCD16-iPSC into effector cells such as induced T cells and induced NK cells. Flow cytometric analysis of A: CAR-hnCD16 iPSC, B: CAR-hnCD16 iCD34 cells, C: CAR-hnCD16-induced T cells. It is shown that activated hnCD16-iNK cells produce soluble factors that enhance T cell activation as measured by the percentage of CD69-positive T cells. It is shown that activated hnCD16-iNK cells produce soluble factors that enhance T cell migration quantified by flow cytometry of T cell migration from the upper chamber to the lower chamber in the transwell assay. It is shown that the induced NK cells enhance the in vivo T cell migration from A: blood to B: peritoneum of the injected mouse model, which is quantified by flow cytometry. Each data point represents an individual mouse. IncuCite ™ real-time imaging of tumor spheroid growth and formation over 84 hours as defined by the applied algorithm mask is shown. A: Induction of SKOV3 spheroid formation during continuous monitoring of changes over time in spheroid size and total integral fluorescence intensity Representative IncuCite ™ imaging of NK cell infiltration, B: T cells alone penetrate into the center of spheroids Although not possible, the addition of induced NK cells promoted T cell infiltration and subsequent spheroid destruction. It is shown that co-expression of IL-15 / IL-15ra (denoted as IL-15RF) enhances in vitro persistence of induced NK cells expressing NK-CAR19 in the absence of soluble IL-2. It is shown that co-culture of iPSC-derived NK enhances T cell infiltration of tumor spheroids by measuring the total integrated fluorescence intensity in the largest red object mask. iPSC-derived NK cells synergize with T cells to enhance the production of (A) TNFα and (B) IFNγ by both CD4 ⁺ and CD8 ⁺ T cells during co-culture with SKOV-3 tumor spheroids. Is shown. We show that engineered CAR-iT cells expressing a high-affinity non-cleavable version of CD16 represent an opportunity for a secondary approach to target tumors and alleviate tumor antigen escape via ADCC. Both CAR and hnCD16 ADCC-mediated cytotoxicity are used for CD19 +/+ and CD19 − / − Raji cells. After 36 hours in the presence and absence of the anti-CD20 monoclonal antibody rituximab, the survival of target cells was quantified by Incute Zoom. It shows cell proliferation of TRAC-CAR-iT cells during the 35-day differentiation process, and initiation of TRAC-CAR TiPSC in a single production run results in a 40,000-fold increase in cell yield. Differentiated synthetic cells were transferred from monolayer to suspension culture on about 28 days. It represents that D35 TRAC-CAR-iT cells were evaluated for the production of pro-inflammatory cytokines IFNg and TNFa, and (B) survival-promoting cytokine IL-2 in response to 4-hour PMA / ionomycin stimulation. (A) Primary CAR-T cells and (B) Synthetic T cells using 18-hour flow cytometry with wild-type (CD19 +/+) or knockout (CD19− / −) NALM-6 as target cells. A comparison of in vitro cytotoxicity between TRAC-CAR-iT is shown. Three independent experiments with three separate primary CAR-T cells and three independent experiments with three separate TRAC-CAR-iT production batches were used. In a transwell migration assay using (A) D20 TRAC-CAR-iT cells and (B) D28 TRAC-CAR-iT cells, TRAC-CAR-iT cells showed chemotaxis in response to thymus-derived chemokines. Indicates that it has been evaluated. In iPSC-derived NK cells expressing hnCD16, anti-CD19 CAR, and IL15 / IL15Ra, enhanced NK cell maturation, (A) increased production of granzyme B, and (B) increased KIR2DL3 and KIR2DL1. Shows the expression. IL15 / IL15RA expression is shown to promote persistence and antigen driven proliferation iNK, the (A) 1 × 10 ⁷ of CAR iNK or CAR-IL15 / IL15Ra iNK cells 0,7, and 14 days Immunodeficient NOD mice were IV injected. IL-2 is the first three weeks are twice IP administration per week, once a 9 week week for sustained evaluation, iNK cells in the blood is measured, the Nalm6 cells (B) ⁵ × 10 5 It was transplanted into NSG mouse IV. After 4 and 11 days, 5 × 10 ⁶ CAR iNK or CAR-IL-15 / IL-15ra iNK was IV injected and the number of iNK cells in the blood was measured weekly by flow cytometry to assess cell proliferation. IL-2 was IP-administered twice a week. (A) CAR-IL15 / IL15Ra iNK cells improved survival in a highly aggressive dissemination model of B-cell lymphoma (p = 0.018, CAR-IL-15 / IL-15ra iNK vs. CAR iNK). ), And (B) CAR-IL15 / IL15Ra iNK cells are shown to prevent tumor progression in vivo in the Nalm6 xenograft model of leukemia. (A) In a 4-hour cytotoxicity assay, hnCD16 relative to NALM6 and NALM6 CD19 ^{− / −} measured using hnCD16-CAR-IL-15 / IL-15ra iNK cells with increased E: T ratio. -CAR-IL15 / IL15Ra Shows in vitro cytotoxicity of iNK cells. Using (B) ARH-77 leukemic cells or (C) ARH-77 CD19 ^{− / −} cells, cytotoxic and rituximab-induced ADCCs were used in a 4-hour cytotoxic assay against unmodified iNK cells. Measured directly. In a mixed culture cytotoxicity assay, we show hnCD16, CAR, and IL-15 / IL-15ra modality that synergize to eradicate the targets of CD19 + and CD19-. Parental ARH-77 cells (CD19 ⁺ ) and ARH-77 CD19 ^- cells were transduced with a red fluorescent tag and a green fluorescent tag, respectively. These cells were mixed 1: 1 and used as target cells in a long-term cytotoxicity assay utilizing various iNK cell populations as effector cells in the presence or absence of rituximab antibody. The frequency of green CD19- and red CD19 + targets was measured throughout the assay using the Incucite imaging system to quantify cytotoxicity to both target cells within a single well. The data are plotted as the frequency of target cells remaining in both target types, normalized to controls without effector cells (tumor cells only). Using a mixed lymphocyte response (MLR) assay, which compares the proliferative response of TRAC-CAR iT cells to primary CAR-T cells against HLA-incompatible PBMC-derived T cells as target cells, TRAC-CAR iT cells Shows that it is not alloreactive to HLA-incompatible healthy cells. Responder cells were labeled with cell trace dye and after 4 days flow cytometry was used to assess dye dilution. We show that TRAC-CAR iT cells expressing hnCD16 are a secondary approach to target tumors. The cytotoxicity to CD19 +/+ and CD19 − / − Raji cells via CAR and hnCD16 ADCC was compared. After 72 hours in the presence and absence of the anti-CD20 monoclonal antibody rituximab, the survival of target cells was quantified by flow cytometry.

Genome modification of iPSCs (induced pluripotent stem cells) includes insertion, deletion, and substitution of polynucleotides. Exogenous gene expression in genome-manipulated iPSCs is gene silencing in dedifferentiated cell types from cells derived from genome-manipulated iPSCs after long-term clonal proliferation of the original genome-manipulated iPSCs, after cell differentiation. We often encounter problems such as silencing or decreased gene expression. On the other hand, direct manipulation of primary immune cells such as T cells or NK cells is difficult and impairs the preparation and delivery of engineered immune cells for adoptive cell therapy. The present invention provides an efficient and reliable targeting approach for the stable integration of one or more exogenous genes, including suicide genes and other functional modalities, which include engraftment, transport, homing and migration. Improves therapeutic properties regarding cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or viability into iPSC-induced cells obtained through indicated iPSC differentiation, this Inducible cells include, but are not limited to, HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells.

Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings commonly understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include the plural and plural terms shall include the singular.

It should be understood that the present invention is not limited to, but may vary, from the particular methodologies, protocols, and reagents described herein. The terminology used herein is for illustration purposes only and is not intended to limit the scope of the invention as defined solely by the claims.

As used herein, the articles "a," "an," and "the" are used herein to refer to one or more of the grammatical objects of the article (ie, at least one). As used herein to refer. As an example, "an element" means one element or more than one element.

The use of alternatives (eg, "or") should be understood to mean either one, both, or any combination thereof.

The term "and / or" should be understood to mean either one or both alternatives.

As used herein, the term "about" or "approximately" is used as compared to the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of a reference. Amount, level, value, number, frequency, percentage, which varies in amounts of%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or even 1%. Refers to dimensions, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" is approximately ± 15%, ± of the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of a reference. Level, value, number, frequency, percentage of 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% , Dimensions, size, quantity, weight, or range of length.

As used herein, the term "substantially" or "essentially" is compared to the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of a reference. And the amount, level, value, number, frequency, which is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more. Refers to percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms "substantially the same" or "essentially the same" are approximately identical to the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of a reference. Refers to a range of reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths.

As used herein, the terms "substantially free" and "essentially free" are used interchangeably and are used to describe compositions such as cell populations or culture media. When, for example, 95% free, 96% free, 97% free, 98% free, 99% free, etc. of a particular substance or source thereof, or measured by conventional means. Refers to a composition that does not contain a particular substance or its source, such as undetectable. The terms "free" or "essentially free" of a particular ingredient or substance in the composition also include (1) no such ingredient or substance in the composition at any concentration, or ( 2) It also means that it is contained in the composition but has a low density and is functionally inactive. A similar meaning can be applied to the term "non-existent" and refers to the absence of a particular substance in the composition or its source.

Throughout this specification, "comprise," "comprises," and "comprising" are the steps, elements, or elements described, unless the context requires otherwise. It will be understood that it is meant to include steps or groups of elements, but not to exclude any other steps or elements, or steps or groups of elements. In certain embodiments, the terms "include," "have," "include," and "include" are used synonymously.

By "consisting of" is meant to include and be limited to those following the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the enumerated elements are required or required and that no other element can exist.

"Consistent essentially of" means to include any element listed after the phrase, which does not interfere with the activity or behavior specified in the disclosure of the listed elements. Or limited to other elements that do not contribute to them. Therefore, the phrase "becomes essential from" indicates that the enumerated elements are necessary or essential, but the other elements are not as needed and may affect the activity or behavior of the enumerated elements. Indicates that it may or may not exist, depending on whether it affects it or not.

Throughout this specification, "one embodiment", "embodiment", "specific embodiment", "related embodiment", "specific embodiment", "additional embodiment", or "further embodiment". "Or a reference to a combination thereof means that a particular feature, structure, or property described in connection with an embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the aforementioned terms in various places throughout the specification does not necessarily refer to the same embodiment. Moreover, specific features, structures, or properties can be combined in any suitable way in one or more embodiments.

The term "ex vivo" is generally performed in vitro, such as experiments or measurements performed in or on living tissue in an in vitro artificial environment, preferably with minimal changes in natural conditions. Refers to an activity. In certain embodiments, the "ex vivo" procedure is taken from the living body and is typically taken from the body under sterile conditions, typically up to several hours or about 24 hours, but up to 48 hours or 72 hours or more depending on the situation. Includes live cells or tissues cultured in an experimental device, including. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures that last longer than a few days using living cells or tissues are typically considered to be "in vitro," but in certain embodiments, the term is ex vivo compatible. Can be used for.

The term "in vivo" generally refers to activities that occur within the body.

As used herein, the terms "reprogramming" or "dedifferentiation" or "increased differentiation potential" or "increased developmental potential" are methods of increasing the differentiation potential of a cell, or better dividing a cell. It refers to a method of dedifferentiation into an undifferentiated state. For example, increased differentiation potential has more developmental plasticity (ie, can differentiate into more cell types) compared to the same cell in the unreprogrammed state. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that is not reprogrammed.

As used herein, the term "differentiation" refers to unspecialized ("uncommitted") or less specialized cells that characterize specialized cells, such as blood cells or muscle cells. The process of acquiring. Differentiated or induced differentiation cells are cells that exhibit more specialized (“committed”) positions within the cell lineage. The term "committed", when applied to the process of differentiation, follows the differentiation pathway to a position where it continues to differentiate into a particular cell type or subset of cell types under normal circumstances and is different under normal circumstances. A cell that cannot differentiate into a cell type or return to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of cells (ie, the embryo itself) to form all lineages of living or somatic cells. For example, an embryonic stem cell is a type of pluripotent stem cell capable of forming cells from each of three germ layers, an ectoderm, a mesoderm, and an endoderm. Pluripotency is more primitive and more capable of developing a complete organism from incomplete or partially pluripotent cells (eg, epiblast stem cells or EpiSC) that cannot produce a complete organism. It is a continuum of developmental potential that extends to pluripotent cells (eg, embryonic stem cells).

As used herein, the term "induced pluripotent stem cells" or iPSC refers to stem cells being generated from induced or altered, differentiated adult, neonatal or fetal cells, i.e. all three. Germ or derm layer: means reprogrammed into cells that can differentiate into all tissues of the germ layer, endoderm, and ectodermal. The iPSCs produced do not refer to naturally occurring cells.

As used herein, the term "embryonic stem cell" refers to a naturally occurring pluripotent stem cell in the inner cell mass of a blastocyst. Embryonic stem cells are pluripotent and generate derivatives of the ectoderm, endoderm, and mesoderm of all three major germ layers. They do not contribute to the extraembryonic membrane or placenta, ie they are not totipotent.

As used herein, the term "pluripotent stem cell" differentiates into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Refers to cells that have. Therefore, pluripotent cells can also be referred to as "partially differentiated cells." Pluripotent cells are well known in the art and examples of pluripotent cells include adult stem cells such as hematopoietic stem cells and neural stem cells. "Popularity" indicates that cells form many types of cells of a particular lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.) but not neurons. Thus, the term "pluripotency" refers to the condition of cells that are totipotent and have a lower degree of developmental potential than pluripotency.

Pluripotency can be determined in part by assessing the pluripotency properties of cells. The characteristics of pluripotency include (i) pluripotent stem cell morphology, (ii) unlimited self-renewal potential, (iii) SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, Pluripotency such as TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominine, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50. Expression of stem cell markers, (iv) ability to differentiate into all three somatic lineages (ectoderm, mesodermal, and endoderm), (v) formation of malformations consisting of three somatic lineages, (vi) 3 It includes, but is not limited to, the formation of germ layer consisting of cells of one somatic cell lineage.

Pluripotency in a "priming" or "quasi-stable" state similar to the blastocyst stem cells (EpiSC) of late blastocysts and "naive" or "naive" similar to the inner cell mass of early / pre-implantation blastocysts Two types of pluripotency have been described so far: pluripotency in the "ground" state. Both pluripotent states exhibit the characteristics described above, while naive or ground states are (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) enhancement during single cell culture. Cloning and viability, (iii) overall reduction in DNA methylation, (iv) reduction in H3K27me3 inhibitory chromatin mark deposition on developmental regulatory gene promoters, (v) compared to primed pluripotent cells It further shows the reduced expression of differentiation markers. The standard methodology for cell reprogramming, in which an exogenous pluripotency gene is introduced into a somatic cell, expressed, and then silenced or removed from the resulting pluripotency cell, is generally pluripotency priming. It is believed to have state characteristics. Under standard pluripotent cell culture conditions, such cells remain in the priming state and ground state characteristics are observed unless exogenous transgene expression is maintained.

As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological features of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a high nuclear-to-cytoplasmic ratio, prominent nucleoli, typical intercellular spacing, rounded and small shape.

As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domestic animal.

A "pluripotency factor" or "reprogramming factor" refers to a drug that can increase the developmental potential of cells alone or in combination with other drugs. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of cells. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

"Culture" or "cell culture" refers to the maintenance, proliferation and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (in each case, a single "medium"), "supplementary component" and "medium supplementary component" refer to a nutritional composition for culturing a cell culture.

"Culturing" or "maintaining" means maintaining, proliferating (proliferating), and / or differentiating cells in tissues or in vitro, for example, in sterile plastic (or coated plastic) cell culture dishes or flasks. Point to. “Culturing” or “maintaining” can utilize the medium as a source of nutrients, hormones, and / or other factors that help the growth and / or maintenance of cells.

As used herein, the term "mesoderm" appears during early embryonic development and includes a variety of circulatory blood cells, muscle, heart, dermis, skeleton, and other supporting and connective tissues. Refers to one of three germ layers that give rise to a specialized cell type.

As used herein, the term "secondary hematopoietic endothelium" (HE) or "pluripotent stem cell-derived secondary hematopoietic endothelium" (iHE) refers to hematopoietic stem cells and progenitor cells in a process called endothelial hematopoietic conversion. Refers to a subset of endothelial cells that give rise to. Development of hematopoietic cells in the embryo progresses sequentially from the lateral plate mesoderia to secondary hematopoietic endothelial cells and hematopoietic progenitor cells via hemangioblasts.

The terms "hematopoietic stem cells and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic progenitor cells" are committed to the hematopoietic lineage, but allow for further hematopoietic differentiation and pluripotency hematopoiesis. Refers to cells containing stem cells (blood cells), bone marrow progenitor cells, macronuclear progenitor cells, erythrocyte progenitor cells, and lymphocyte progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) include myeloid lines (monocytes and macrophages, neutrophils, basal cells, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymph lines (T cells, B). It is a pluripotent stem cell that produces all blood cell types, including cells (NK cells). As used herein, the term "secondary hematopoietic stem cells" refers to CD34 + hematopoietic cells that can give rise to both mature myelogenous and lymphoid cell types, including T cells, NK cells, and B cells. Point to. Hematopoietic cells also contain various subsets of primordial hematopoietic cells that give rise to primordial red blood cells, megakaryocytes, and macrophages.

As used herein, the terms "T lymphocytes" and "T cells" are used interchangeably to complete maturation in the thymus, identify specific foreign antigens in the body, and other immune cells. Refers to the major types of leukocytes that have various roles in the immune system, including activation and inactivation of. T cells can be any T cell, eg, cultured T cell, eg, primary T cell, or cultured T cell line, eg, T cell from Jurkat, SupT1, etc., or T cell obtained from a mammal. .. T cells can be CD3 + cells. T cells include CD4 + / CD8 + double positive T cells, CD4 + helper T cells (eg, Th1 and Th2 cells), CD8 + T cells (eg, cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes. Any type of T cell, including, but not limited to, (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), and the like. Can be of any developmental stage. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express the T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

"CD4 + T cells" refers to a subset of T cells that express CD4 on their surface and are associated with a cell-mediated immune response. They are characterized by a post-stimulation secretory profile and can include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen for T lymphocytes, but is also found in other cells containing monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as a related cognitive element of the MHC (major histocompatibility complex) class II binding immune response. For T lymphocytes, we define a subset of helpers / inducers.

"CD8 + T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I constrained, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found in thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a related recognition element of major histocompatibility complex class I binding interactions.

As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). .. As used herein, the terms "adaptive NK cells" and "memory NK cells" are compatible, phenotypically CD3- and CD56 +, at least one of NKG2C and CD57, and Refers to a subset of NK cells that express CD16 as needed, but lack the expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated subpopulation of CD56 + NK cells is CD16, NKG2C, CD57, NKG2D, NCR ligand, NKp30, NKp40, NKp46, activated and inhibitory KIR, NKG2A, and / or DNAM-1. Including the expression of. CD56 + can be a dark or bright expression.

As used herein, the term "NKT cell" or "natural killer T cell" refers to a CD1d-binding T cell that expresses the T cell receptor (TCR). Unlike traditional T cells, which detect peptide antigens presented by traditional major histocompatibility complex (MHC) molecules, NKT cells recognize lipid antigens presented by the nonclassical MHC molecule CD1d. Two types of NKT cells are recognized. Invariants or type I NKT cells express a very limited TCR repertoire-a regular α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant Type II NKT cells, presents a more heterogeneous use of TCRαβ. Type I NKT cells are considered suitable for immunotherapy. Adapted or invariant (type I) NKT cells can be identified by expression of at least one of the markers TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

As used herein, terms such as "isolated" have been isolated from their original environment, i.e. the environment of the isolated cells is "unisolated" reference cells present. Refers to a cell or population of cells that is substantially free of at least one component found in the environment. The term includes, for example, cells isolated from a tissue or biopsy sample and taken from some or all components as found in its natural environment. The term also includes cells taken from at least one, some, or all components because the cells are found in a non-natural environment, eg, an environment isolated from a cell culture or cell suspension. .. Thus, isolated cells, when found in nature or when grown, stored, or survive in a non-natural environment, are partially or completely from at least one component, including other substances, cells, or cell populations. Is separated into. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in non-naturally occurring media. An isolated cell can be obtained from separating the desired cell or population thereof from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment. it can.

As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

As used herein, the term "coding" refers to a defined sequence of nucleotides (ie, rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting from them. Refers to the unique properties of a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that serves as a template for the synthesis of other polymers and macromolecules in a biological process that has. Thus, if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system, the gene encodes the protein. Both the coding strand, which has the same nucleotide sequence as the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be a protein or other of that gene or cDNA. It can be said that it encodes a product.

"Construct" refers to a macromolecular or molecular complex containing a polynucleotide delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can induce delivery or introduction of a foreign genetic material into a target cell and can replicate and / or express in the target cell. .. As used herein, the term "vector" includes the construct to be delivered. The vector can be a linear or cyclic molecule. Vectors may or may not be incorporated. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

By "integration" is meant that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently attached to a nucleic acid sequence in the chromosomal DNA of the cell. By "targeted integration" is meant that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a preselected site or "integration site". As used herein, the term "integration" includes the insertion of one or more exogenous sequences or nucleotides of a construct with or without deletion of an endogenous sequence or nucleotide at the site of integration. Refers further to the process. If there is a deletion at the insertion site, "integration" may further include substitution of nucleotides deleted with an endogenous sequence or one or more inserted nucleotides.

As used herein, the term "exogenous" is intended to mean that a reference molecule or reference activity is introduced into or unnatural to a host cell. There is. The molecule can be introduced, for example, by introducing the coding nucleic acid into the genetic material of the host, eg, by integrating into the host chromosome, or as a non-chromosomal genetic material, eg, a plasmid. Therefore, the term used with respect to the expression of a coding nucleic acid refers to the introduction of the coding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in a host cell. Similarly, the term refers to the expression of a coding nucleic acid that is contained intracellularly and has not been exogenously introduced when used with respect to the expression of the coding nucleic acid.

As used herein, a "gene of interest" or "polynucleotide sequence of interest", when placed under the control of an appropriate regulatory sequence, is transcribed into RNA and, in some cases, into a polypeptide in vivo. The DNA sequence to be translated. Genes or polynucleotides of interest may include, but are limited to, prokaryotic sequences, cDNAs from eukaryotic mRNAs, genomic DNA sequences from eukaryotic (eg, mammalian) DNA, and synthetic DNA sequences. Not done. For example, the gene of interest is a miRNA, shRNA, a naturally occurring polypeptide (ie, a naturally occurring polypeptide) or a fragment thereof, a variant polypeptide (ie, a naturally occurring polypeptide having less than 100% sequence identity. It can encode a variant of a polypeptide) or a fragment thereof, a modified polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

As used herein, the term "polynucleotide" refers to nucleotides of any length in polymer form, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of the polynucleotide consists of four nucleotide bases: cyanedin (A); cytosine (C); guanine (G); thymine (T); and uracil (U) instead of thymine when the polynucleotide is RNA. .. Polynucleotides include genes or gene fragments (eg, probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNAs (mRNAs), transfer RNAs, ribosome RNAs, ribozymes, cDNAs, recombinant polynucleotides, branched strands. It can include polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to molecules that have amino acid residues covalently linked by peptide bonds. The polypeptide must contain at least two amino acids and there is no limit to the maximum number of amino acids in the polypeptide. As used herein, these terms refer to, for example, both short chains, commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, commonly referred to in the art as polypeptides or proteins. .. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives. , Equivalents, fusion proteins are included. Polypeptides include native polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

"Operably linked" refers to the binding of nucleic acid sequences on a single nucleic acid fragment, where one function is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of its coding sequence or functional RNA (ie, the coding sequence or functional RNA is transcription of the promoter). Under control). The coding sequence can be operably linked to the regulatory sequence in the sense or antisense orientation.

As used herein, the term "gene imprint" contributes to the preferred therapeutic attributes of source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived epigenetic cells. Refers to genetic or epigenetic information. As used herein, "source cells" are non-pluripotent cells that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs are specific, including any hematopoietic lineage cells. Can further differentiate into cell types of. IPSCs derived from source cells, and cells differentiated from them, may be collectively referred to as "inducible" cells or "inducible" cells, depending on the context. For example, inducible effector cells, or inducible NK cells or inducible T cells, used throughout this application are their primary counterparts obtained from natural / natural sources such as peripheral blood, cord blood, or other donor tissues. When compared with the thing, it is a cell differentiated from iPSC. As used herein, genetic imprints that impart preferential therapeutic attributes are by reprogramming selected source cells that are specific to the donor, disease, or therapeutic response, or genome editing. By introducing the recombinant modality into the iPSC using the above, it is incorporated into the iPSC. In the aspect of the source cell obtained from a specific selected donor, disease, or therapeutic context, whether the underlying molecular event is identified in the genetic imprint that contributes to the preferred therapeutic attribute. Regardless, it may include a retentive phenotype, ie, a context-specific genetic or epigenetic, representing a preferred therapeutic attribute, which is passed to the inducing cells of the selected source cell. Source cells specific for donor, disease, or therapeutic response can include gene imprints that can be retained by iPSCs and derived hematopoietic cells, which include virus-specific T cells or imprints. Pre-positioned unispecific TCRs from variant natural killer T (iNKT) cells, traceable desirable genetic polymorphisms, eg, homozygous point mutations encoding high affinity CD16 receptors of selected donors Includes, but is not limited to, connectivity, predetermined HLA requirements, i.e., selected HLA-matched donor cells that exhibit an increased population of haplotypes. As used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, regulation and regulation of immune response, viability, of induced cells. And cytotoxic. Preferred therapeutic attributes were also improved: expression of antigen-targeted receptors, presentation or lack of HLA, resistance to tumor microenvironment, induction and immunomodulation of bystander immune cells, reduced extratumor effects. It may also be associated with target specificity and resistance to treatments such as chemotherapy.

As used herein, the term "enhanced therapeutic properties" refers to the therapeutic properties of enhanced cells as compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties as compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells include, but are not limited to, cell engraftment, transport, homing, viability, self-renewal, persistence, regulation and regulation of immune responses, viability, and cytotoxicity. The therapeutic properties of immune cells include expression of antigen-targeted receptors, presentation or lack of HLA, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved targets with reduced extratumor effects. It is also manifested by specificity and resistance to treatments such as chemotherapy.

As used herein, the term "engager" forms a link between immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, neutrophils, and tumor cells. Refers to a molecule capable of activating immune cells, such as a fusion polypeptide. Examples of engagers include bispecific T cell engagers (BiTE), bispecific killer cell engagers (BiKE), trispecific killer cell engagers, or multispecific killer cell engagers, or multiple. Includes, but is not limited to, universal killer cells that may be compatible with the immune cell type of.

As used herein, the term "surface-triggered receptor" refers to a receptor that can elicit or initiate an immune response, such as a cytotoxic response. Surface-triggered receptors can be engineered and expressed on effector cells such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, the surface-triggered receptor is a bispecific antibody or multiplex between the effector cell and a specific target cell, such as a tumor cell, regardless of the natural receptor and cell type of the effector cell. Promotes binding of specific antibodies. Using this approach, iPSCs containing universal surface-triggered receptors can be generated and such iPSCs can be differentiated into a population of various effector cell types expressing universal surface-triggered receptors. "Universal" means that surface-triggered receptors can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor are subject to the tumor binding specificity of the engager. Regardless, it can bind or bind to an engager with the same epitope recognized by the surface-triggered receptor. In some embodiments, an engager with the same tumor targeting specificity is used to bind to the universal surface trigger receptor. In some embodiments, engagementrs with different tumor targeting specificities are used to bind to universal surface trigger receptors. Therefore, one or more effector cell types may be used to kill specific types of tumor cells, or two or more types of tumors. Surface-triggered receptors generally contain a costimulatory domain for activation of effector cells and an anti-epitope specific to the epitope of an engager. The bispecific engager is specific for the anti-epitope of the surface-triggered receptor at one end and the tumor antigen at the other end.

As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent the potential toxicity or other adverse effects of cell therapy. In some examples, the expression of the safety switch protein is conditionally regulated, addressing safety concerns of transplanted engineered cells that permanently integrate the gene encoding the safety switch protein into the genome. This conditional regulation can vary and may include small molecule-mediated post-translational activation and tissue-specific and / or transient transcriptional regulation. Safety switches can mediate apoptosis induction, protein synthesis inhibition, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation and / or antibody-mediated depletion. In some examples, safety switch proteins are activated by exogenous molecules, such as prodrugs, and when activated, induce apoptosis and / or cell death in therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as, but not limited to, caspase-9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. .. In this strategy, the prodrug administered at the time of the occurrence of an adverse event is activated by the suicide gene product, killing the transduced cells.

As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide that can achieve biological and / or pharmaceutical effects on an organism. Pharmaceutically active proteins have curative or palliative properties for the disease and can be administered to ameliorate, alleviate, alleviate, reverse or alleviate the severity of the disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the development of a disease or to reduce the severity of such a disease or morbidity when it appears. .. Pharmaceutically active proteins include whole proteins or peptides or pharmaceutically active fragments thereof. It also includes pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term pharmaceutically active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines. ..

As used herein, the term "signal transduction molecule" refers to any molecule that regulates, engages, inhibits, activates, reduces, or increases cell signaling. Signal transduction refers to the transmission of molecular signals in the form of chemical alterations by the recruitment of protein complexes along the pathways that ultimately cause biochemical events within the cell. Signal transduction pathways are well known in the art and are G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, tallgate signaling, ligand-dependent ion channel signaling, ERK / MAPK signaling pathways. , Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways, but are not limited to these.

As used herein, the term "targeted monoclonality" is used when i) is genetically integrated into a cell and is associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR). Antigen specificity, ii) Engager specificity when associated with monoclonal antibodies or bispecific engagers, iii) Targeting transformed cells, iv) Targeting cancer stem cells, and v) Specific antigens or Refers to molecules that promote the specificity of antigens and / or epitopes, including, but not limited to, other targeting strategies in the absence of surface molecules, such as polypeptides.

As used herein, the term "specific" or "specificity" refers to a molecule that selectively binds to a target molecule, eg, a receptor, as opposed to non-specific or non-selective binding. Or it can be used to refer to the ability of an engager.

As used herein, the term "adoptive cell therapy" is identified as T or B cells that are ex vivo proliferating, genetically modified, or unmodified prior to transfusion. Refers to cell-based immunotherapy as used herein, which is associated with transfusion of autologous or allogeneic lymphocytes.

As used herein, "therapeutically sufficient amount" is, within its meaning, non-toxic but sufficient and / or effective for the particular therapeutic and / or pharmaceutical composition it refers to. Including the amount to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the illness and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, alleviate, and / or ameliorate at least one symptom associated with the disease or condition of the subject being treated.

Differentiation of pluripotent stem cells requires changes in the culture system, such as changes in the stimulant in the culture medium and the physical state of the cells. The most common strategy utilizes the formation of the embryoid body (EB) as a common and important intermediate for initiating lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development, generating a large number of lineages within a three-dimensional region. Through a differentiation process, typically hours to days, simple EBs (eg, differentiation-induced aggregate pluripotent stem cells) continue to mature and grow into cystic EBs, typically from days to days. At this time, which is several weeks, they are processed to continue to differentiate. EB formation is initiated by bringing pluripotent stem cells close to each other within a three-dimensional multi-layer cluster of cells, which typically precipitates pluripotent cells as droplets and "U" bottom wells of cells. It is achieved by one of several methods, including settling on a plate or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form suitable EBs, so pluripotent stem cell aggregates require further differentiation clues to promote EB growth. Therefore, pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides cue induction to selected strains. EB-based cultures of pluripotent stem cells typically produce moderately proliferated, differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) within EB cell clusters. Although EB has been shown to promote cell differentiation, the inconsistent exposure of cells of three-dimensional structure to the queue of differentiation from the environment gives rise to heterologous cells in a variety of differentiated states. In addition, EBs are cumbersome to create and maintain. Furthermore, cell differentiation by EB is accompanied by appropriate cell proliferation, leading to a decrease in differentiation efficiency.

In contrast, "aggregate formation", unlike "EB formation", can be used to proliferate a population of pluripotent stem cell-derived cells. For example, during the growth of aggregate-based pluripotent stem cells, the culture medium is selected to maintain growth and pluripotency. Cell proliferation generally increases the size of aggregates that form larger aggregates, which routinely mechanically or enzymatically dissociate into smaller aggregates to maintain cell proliferation in culture. And the number of cells can be increased. Unlike EB cultures, cells cultured in aggregates in maintenance cultures maintain pluripotency markers. Pluripotent stem cell aggregates require a further differentiation queue to induce differentiation.

As used herein, "monolayer differentiation" is a term that refers to a method of differentiation that differs from differentiation by three-dimensional multi-layer clusters of cells, i.e., "EB formation." Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Since monolayer culture does not mimic embryogenesis such as EB formation, differentiation into a particular lineage is considered minimal compared to germ layer differentiation of all three EBs.

As used herein, "dissociated" cells refer to cells that have been substantially isolated or purified from other cells or from the surface (eg, the surface of a culture plate). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other, for example, by enzymatic or mechanical dissociation into a suspension of clusters, a single cell or a mixture of single cells and clusters. In yet another alternative embodiment, the adherent cells are dissociated from the culture plate or other surface. Thus, dissociation involves disrupting cellular interactions with extracellular matrix (ECM) and substrates (such as culture planes), or disrupting ECM between cells.

As used herein, a "feeder cell" or "feeder" is co-cultured with a second type cell to provide an environment in which the second type cell can proliferate, proliferate, or differentiate. A term used to describe one type of cell, feeder cells provide stimuli, growth factors, nutrients, and support a second type of cell. Feeder cells may be derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary culture of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the proliferation and maturation of natural killer cells. Feeder cells are typically treated with irradiation or an anti-mitotic agent such as mitomycin to prevent them from proliferating more than the cells they support when co-cultured with other cells. Can be inactivated. Feeder cells can include endothelial cells, stromal cells (eg, epithelial cells or fibroblasts), and leukemia cells. Without limiting the above, one particular feeder cell type can be a human feeder such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, some of the various feeder cells are used to maintain pluripotency, direct differentiation to specific lineages, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells. be able to.

As used herein, a "feeder-free" (FF) environment is a culture condition, cells that are essentially free of feeders or stromal cells and / or have not been pretreated by culturing feeder cells. Refers to an environment such as culture or culture medium. "Pretreated" medium refers to medium collected after feeder cells have been cultured in the medium for a period of time, such as at least one day. The pretreated medium contains many mediator substances, including growth factors and cytokines secreted from feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain both feeder cells or stromal cells and has not been pretreated by culturing the feeder cells.

"Functional" as used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated from them, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed from them. Successful knock-in, knock-out, at the genetic level, achieved by (1) direct genome editing or modification, or by "passage" through differentiation or reprogramming from the first genome-engineered starting cell. Knockdown gene expression, transgenic or controlled gene expression, such as inducible or transient expression at the desired cell development stage, or (2) (i) obtained in the above cells by direct genome editing. Modification of gene expression, (iii) modification of gene expression maintained in the cells by "passage" through differentiation or reprogramming from the first genome-engineered starting cell, (iii) at the early developmental stage of the cells. Genetic regulation downstream of the cells, or (iv) iPSC, progenitor cells, or dedifferentiated as a result of gene expression alterations that only appear or appear only in the initiating cells that give rise to the cells via differentiation or reprogramming. Removal, addition, or cellular function / property at the cellular level due to the enhanced or newly achieved cellular function or attributes presented within the mature cell product, originally derived from genomic editing or modification made of cellular origin. Refers to the change of.

An "HLA deficiency" that includes an HLA class I deficiency, an HLA class II deficiency, or both is the surface of a complete MHC complex that contains an HLA class I protein heterodimer and / or an HLA class II heterodimer. A cell whose level of expression is deficient or no longer maintained, or whose reduced, reduced or reduced level is lower than that naturally detectable by other cells or synthetic methods.

As used herein, "modified HLA-deficient iPSC" refers to improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, etc. Cytokine stimulation, cytokine production (autoclin or paraclin), chemotactic, and cytotoxic, such as non-classical HLA class I proteins (eg, HLA-E and HLA-G), chimeric antigen receptor (CAR), Expresses proteins related to, but not limited to, T cell receptor (TCR), CD16 Fc receptor, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1. Refers to an HLA-deficient iPSC that is further modified by the introduction of a gene. "Modified HLA-deficient" cells also include cells other than iPSC.

"Fc receptor" is abbreviated as FcR and is classified based on the type of antibody recognized. For example, those that bind to the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE. Is called the Fc-epsilon receptor (FcεR). The classes of FcR are also distinguished by the signaling properties of the cells expressing them (macrophages, granulocytes, natural killer cells, T and B cells) and each receptor. Some Fc-gamma receptors (FcγR) have different antibody affinities due to their different molecular structures, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b). Includes members of

The "chimeric Fc receptor," abbreviated as CFcR, is engineered in which the natural transmembrane and / or intracellular signaling domain has been modified or replaced by a non-natural transmembrane and / or intracellular signaling domain. A term used to describe a Fc receptor. In some embodiments of the chimeric Fc receptor, in addition to one or both of the transmembrane and signaling domains being unnatural, one or more stimulation domains are applied to the intracellular portion of the engineered Fc receptor. It can be introduced to enhance cell activation, proliferation, and function by inducing receptors. Unlike a chimeric antigen receptor (CAR) that contains an antigen-binding domain to a target antigen, the chimeric Fc receptor binds to an Fc fragment, or Fc region of an antibody, or an Fc region contained in an engager or binding molecule. It binds to molecules and activates cell functions with or without the target cells in close proximity. For example, the Fcγ receptor is engineered to include selected transmembrane domains, stimulation domains, and / or signaling domains in the intracellular region that responds to IgG binding in the extracellular domain and thereby produces CFcR. Can be done. In one example, a CFcR is produced by manipulating the Fcγ receptor CD16 by replacing its transmembrane domain and / or intracellular domain. To further enhance the binding affinity of CD16-based CFcR, an extracellular domain of CD64 or a high affinity variant of CD16 (eg, F176V) can be incorporated. In some embodiments of CFcR comprising a high affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or the extracellular domain of the receptor appears to be non-cleavage. Is replaced by, i.e., not shed, which results in an hnCD16-based CFcR.

It has been confirmed that the FcγR receptor CD16 has two isoforms, the Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activates NK cells, and promotes antibody-dependent cellular cytotoxicity (ADCC). As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16 (hnCD16)" refers to a natural or non-natural variant of CD16. Wild CD16 has a low affinity and when NK cells are activated, it is subjected to external domain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants with altered or deleted cleavage sites (positions 195 to 198) in the proximal membrane region (positions 189 to 212) are not shed. The cleavage site and proximal membrane region are described in detail in WO2015148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. The CD16 variant containing both F158V and S197P has high affinity and is non-cleavable. Another exemplary high affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 containing an external domain derived from one or more of the three exxons of the CD64 external domain.

I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties Provided herein are the ability of iPSCs to differentiate and the cell development of iPSCs and their induced cells while enhancing the therapeutic properties of the induced cells. It is a strategy to systematically operate the control circuit of the cloned iPSC without affecting the science. Inducible cells have been functionally improved and are suitable for adoptive cell therapy after a combination of selective modality has been introduced into cells at the iPSC level through genomic engineering. Prior to the present invention, the modified iPSCs, including one or more gene edits provided, were capable of still entering cell development while retaining modified activity, and / or producing mature and functional differentiated cells. It was unclear if they had the ability to do so. Unexpected failures during directed cell differentiation from iPSCs include specific gene expression or lack thereof at the developmental stage, HLA complex presentation requirements, protein shedding of introduced surface expression modalities, and cell phenotype and / Or due to aspects including, but not limited to, the need for reconstruction of the differentiation protocol that allows for functional changes. The present application states that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that engineered iPSC-derived functional effector cells become effector cells after iPSC differentiation. It has been shown to have enhanced and / or acquired therapeutic properties due to retained, individual or combined genomic alterations.

1. 1. The hnCD16 knock-in CD16 has been identified as two isoforms, the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activates NK cells, and promotes antibody-dependent cellular cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16" refers to various CD16 variants. Wild CD16 has a low affinity and when NK cells are activated, it is subjected to external domain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an exemplary non-cleavable version of CD16 that has been genetically engineered. .. Manipulated CD16 variants, including both F176V and S197P, have high affinity and are non-cleavable, which is described in detail by WO2015 / 148926, the full disclosure of which is available by reference. Incorporated into the specification. In addition, a chimeric CD16 receptor in which the external domain of CD16 is essentially replaced by at least a portion of the external domain of CD64 can also achieve the desired high affinity and irreversible function of the CD16 receptor capable of performing ADCC. In some embodiments, the replacement external domain of the chimeric CD16 comprises one or more of EC1, EC2, and EC3 exxons of CD64 (UniPROtKB_P12314 or an isoform or polymorphic variant thereof).

Thus, in some embodiments, the high affinity non-cleavable CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments it comprises F176V with the cleavage region removed. In some other embodiments, hnCD16 is an exemplary sequence, at least 50%, 55%, 60% when compared to any of SEQ ID NOs: 1-3, each containing at least a portion of the CD64 external domain. , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage of sequences in between. SEQ ID NOs: 1 to 3 are encoded by exemplifying SEQ ID NOs: 4 to 6, respectively. As used herein and throughout this application, the percent identity between the two sequences takes into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Thus, it is a function of the number of identical positions shared by the array (ie,% identity = number of identical positions / total number of positions x 100). Sequence comparisons and determination of the percentage of identity between two sequences can be performed using mathematical algorithms recognized in the art.
SEQ ID NO: 1
(CD64 domain-based construction of 340 amino acids; CD16TM; CD16ICD)
SEQ ID NO: 2
(Construction of CD64 exon-based 336 amino acids; CD16TM; CD16ICD)
SEQ ID NO: 3
(Construction of CD64 exon-based 335 amino acids; CD16TM; CD16ICD)
SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID NO: 6

Therefore, what is provided herein is a clone genetically engineered to contain a high affinity non-cleavable CD16 receptor (hnCD16), among other edits intended and described herein. The iPSC, which is a genetically engineered iPSC, can differentiate into effector cells containing hnCD16 introduced into the iPSC. In some embodiments, the inducible effector cell containing hnCD16 is an NK cell. In some embodiments, the inducible effector cell containing hnCD16 is a T cell. The exogenous hnCD16 expressed in iPSC or its inducing cells is bispecific, trispecific, or multispecific that recognizes the extracellular binding domain of CD16 or CD64 of hnCD16 as well as ADCC antibody or fragment thereof. It also shows high affinity for binding to engagers or binders. Bispecific, trispecific, or multispecific engagers or binders are described further below in this application (see Section I.6). Thus, the present application relates to the extracellular domain of hnCD16 expressed on inducible effector cells in an amount sufficient for therapeutic use in the treatment of conditions, diseases, or infections further detailed in Section V below. An inducible effector cell or a cell population thereof in which one or more preselected ADCC antibodies are preloaded via high affinity binding is provided, and the hnCD16 is a cell of CD64 or a cell of CD16 having F176V and S197P. Includes outer binding domain.

In some other embodiments, the natural CD16 transmembrane domain and / or intracellular domain of hnCD16 is a chimeric Fc receptor (CFCR) non-natural transmembrane domain, non-natural stimulating domain, and / or It is further modified or replaced so that it is generated to include an unnatural signaling domain. As used herein, the term "non-natural" means that the transmembrane domain, stimulus domain, or signaling domain is derived from a different receptor other than the receptor that provides the extracellular domain. In the examples herein, a CFcR based on CD16 or a variant thereof does not have a transmembrane domain, stimulation domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR is CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, Derived from ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T cell receptor polypeptide Includes non-natural transmembrane domains. In some embodiments, the exogenous hnCD16-based CFcR is CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D. Contains a non-natural stimulus / inhibitory domain derived from a polypeptide. In some embodiments, the exogenous hnCD16-based CFcR is CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D poly. Contains a non-natural signaling domain derived from the peptide. In one embodiment of hnCD16, the provided chimeric receptor is a transmembrane domain and a signaling domain from which both are derived from one of the IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides. including. One particular embodiment of the hnCD16-based chimeric Fc receptor comprises a transmembrane domain of NKG2D, a stimulation domain of 2B4, and a signaling domain of CD3ζ, and the extracellular domain of hnCD16 is the extracellular domain of CD64 or CD16. The extracellular domain of CD16 comprises F176V and S197P, derived from the full or partial sequence of. Another embodiment of the hnCD16-based chimeric Fc receptor comprises a transmembrane domain and a signaling domain of CD3ζ, the extracellular domain of hnCD16 being derived from the full or partial sequence of the extracellular domain of CD64 or CD16, CD16. The extracellular domain of is F176V and S197P.

Various embodiments of the hnCD16-based chimeric Fc receptor as described above can be applied to the Fc region of an antibody or fragment thereof, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Can be bound to with high affinity. Upon binding, the stimulatory and / or signaling domain of the chimeric receptor allows activation of effector cells and cytokine secretion, and has an antibody, or tumor antigen-binding component and Fc region described above, bispecific, Trispecific or multispecific engagers or binders kill targeted tumor cells. Without being limited by theory, the CFcR is responsible for the killing ability of effector cells through the unnatural transmembrane, stimulation and / or signaling domains of hnCD16-based chimeric Fc receptors, or through the binding of engageers to external domains. Contribute and increase the proliferation and / or proliferation potential of effector cells. Antibodies and engagers can bring antigen-expressing tumor cells closer to CFcR-expressing effector cells, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, multispecific engagers or binders include B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44. , CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP , MICA / B, PSMA, PAMA, P-cadherin, and ROR1, but not limited to these. Several non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding hnCD16-based CFcR-expressing effector cells when attacking tumor cells Includes CD16 (or CD64) -CD30, CD16 (or CD64) -BCMA, CD16 (or CD64) -IL15-EPCAM, and CD16 (or CD64) -IL15-CD33.

Unlike the endogenous CD16 receptor expressed by primary NK cells that are cleaved from the cell surface following NK cell activation, the non-cleavable version of CD16 of the induced NK avoids shedding of CD16 and is constant. Maintain expression. In induced NK cells, non-cleavage CD16 increases the expression of TNFα and CD107a, which are indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC) and binding of double, triple, or multispecific engagers. ADCC is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells. The additional high affinity properties of hnCD16 introduced into induced NK cells also allow in vitro loading of ADCC antibody into NK cells via hnCD16 prior to administration of the cells to subjects in need of cell therapy. .. As presented, the hnCD16 may comprise F176V and S197P in some embodiments, or comprises a complete or partial external domain derived from CD64 as exemplified by SEQ ID NO: 1, 2, or 3. It may further comprise at least one of a obtained or non-natural transmembrane domain, a stimulating domain, and a signaling domain. As disclosed, the application also applies one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as detailed in Section V below. Provides preloaded, induced NK cells or cell populations thereof.

Unlike primary NK cells, mature T cells from primary sources (ie, natural / natural sources such as peripheral blood, cord blood, or other donor tissues) do not express CD16. Functional T cells in which the iPSC containing the expressed exogenous non-cleavable CD16 can not only express the exogenous CD16 but also perform its function through the gained ADCC mechanism without impairing the developmental biology of the T cell. It was unexpected that it could be differentiated into. This ADCC acquired in induced T cells is double-targeted and / or expressed by reduced or absent CAR-T target antigen, which is common in CAR-T cell therapy, or recognition by CAR (chimeric antigen receptor). Can be further used as an approach to rescue antigen escape, where tumors recur with mutant antigens that avoid. When the induced T cells contain ADCC acquired via exogenous CD16 expression and the antibody targets a tumor antigen different from the tumor antigen targeted by CAR, the antibody is used to rescue the CAR-T antigen escape. However, the recurrence or recurrence of the target tumor, which is often seen in CAR-T treatment, can be reduced or prevented. Such a strategy of reducing and / or preventing antigen escape while achieving dual targeting is similarly applicable to NK cells expressing one or more CARs. The various CARs that can be used in this antigen escape reduction and prevention strategy are described in detail below.

Thus, the present invention provides induced T cells containing exogenous CD16. In some embodiments, the hnCD16 contained in the induced T cells comprises F176V and S197P. In some other embodiments, the hnCD16 contained in the induced T cell comprises a complete or partial external domain derived from CD64 as exemplified by SEQ ID NO: 1, 2, or 3, or is unnatural. It may further include at least one of a transmembrane domain, a stimulation domain, and a signaling domain. As described, such induced T cells have an acquired mechanism of targeting tumors with monoclonal antibodies meditated by ADCC to enhance the therapeutic effect of the antibody. As disclosed, the application also applies one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as detailed in Section V below. Provides preloaded, induced T cells or cell population thereof.

2. 2. Expression of CAR Applicable to genetically engineered iPSCs and their inducible effector cells can be any CAR design known in the art. A CAR, or chimeric antigen receptor, is a fusion protein that generally comprises an external domain that includes an antigen recognition region, a transmembrane domain, and an internal domain. In some embodiments, the external domain can further comprise a signal peptide or leader sequence and / or spacer. In some embodiments, the endodomain can further comprise a signaling peptide that activates effector cells expressing CAR. In some embodiments, the antigen recognition domain is capable of specifically binding to the antigen. In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease or pathogen. In some embodiments, the disease-related antigen is a tumor antigen, and the tumor may be a liquid tumor or a solid tumor. In some embodiments, the CAR is suitable for activating either T cells or NK cells that express the CAR. In some embodiments, the CAR is NK cell specific, including an NK specific signaling component. In certain embodiments, the T cells are derived from CAR-expressing iPSC and the induced T cells are T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, It may include γδ T cells, or a combination thereof. In certain embodiments, the NK cells are derived from CAR-expressing iPSC.

In certain embodiments, the antigen recognition region comprises a mouse antibody, a human antibody, a humanized antibody, camel Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. .. Non-limiting examples of antibody fragments include Fab, Fab', F (ab) '2, F (ab) '3, Fv, Single Chain Antigen Binding Fragment (scFv), (scFv) ₂ , Disulfide Stabilization. Fv (dsFv), minibody, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAb, nanobodies), recombinant heavy chain-only antibodies (VHH), and others that maintain binding specificity across antibodies. Contains antibody fragments of. Non-limiting examples of antigens that can be targeted by CAR include ADGRE2, carbonate dehydration enzyme IX (CALX), CCR1, CCR4, cancer fetal antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA), CDS, CLEC12A, cytomegalovirus (CMV) infection Cellular antigens (eg, cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3 4, EGFIR, EGFR-VIII, ERBB folic acid binding protein (FBP), fetal acetylcholine receptor (AChR), folic acid receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epithelial growth factor receptor 2 (HER-2), human telomerase reverse transcription enzyme (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, Mutin 1 (Muc-) 1), Mutin 16 (Muc-16), Mesocerin (MSLN), NKCSI, NKG2D ligand, c-Met, Cancer-testid antigen NY-ESO-1, Cancer fetal antigen (h5T4), PRAME, Prostatic stem cell antigen (PSCA) ), PRAME prostate-specific membrane antigen (PSMA), tumor-related glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1) , And various pathogenic antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

In some embodiments, the transmembrane domain of CAR is CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM- 1. Natural or modified CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T cell receptor polypeptide. Includes the full length or at least a portion of the transmembrane region.

In some embodiments, the signaling peptides of the intracellular domain (or intracellular domain) are CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, Includes the full length or at least a portion of the polypeptide of NKG2C, or NKG2D. In one embodiment, the CAR signaling peptide is at least about 85%, about 90%, about 95%, about 96%, relative to at least one ITAM (immune receptor tyrosine-based activation motif) of CD3ζ. Includes amino acid sequences that are about 97%, about 98%, or about 99% identical.

In certain embodiments, the internal domain further comprises at least one co-stimulating signaling region. The co-stimulation signaling region is CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof. It can include the full length or at least a portion of the polypeptide. In one embodiment, the cell-applicable CARs provided in this application are at least about 85% about 90%, about 95%, about 96%, relative to the co-stimulating domain derived from CD28, and SEQ ID NO: 7. Includes a signaling domain containing the native or modified ITAM1 of CD3ζ represented by an amino acid sequence of about 97%, about 98%, or about 99% identity. In a further embodiment, the CAR containing the co-stimulating domain derived from CD28 and the natural or modified ITAM1 of CD3ζ also comprises a hinge domain and a transmembrane domain derived from CD28, and the scFv is connected to the transmembrane domain via a hinge. Obtained, the CAR comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 8.
SEQ ID NO: 7
(CD28 co-stimulation of 153 amino acids + CD3ζITAM)
SEQ ID NO: 8
(CD28 hinge of 219 amino acids + TM of CD28 + co-stimulation of CD28 + CD3ζITAM)

In another embodiment, the cell applicable CARs provided in this application are at least about 85% about 90% relative to the transmembrane domain from NKG2D, the co-stimulation domain from 2B4, and SEQ ID NO: 9. Includes a signaling domain containing a natural or modified CD3ζ represented by an amino acid sequence of about 95%, about 96%, about 97%, about 98%, or about 99% identity. The CAR, which comprises a transmembrane domain derived from NKG2D, a co-stimulating domain derived from 2B4, and a signaling domain containing a natural or modified CD3ζ, may further comprise a CD8 hinge, and the amino acid sequence of such structure It is at least about 85% about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 10.
SEQ ID NO: 9
(263 amino acids, NKG2D TM + 2B4 + CD3ζ)
SEQ ID NO: 10
(308 amino acids, CD8 hinge + NKG2D TM + 2B4 + CD3ζ)

Non-limiting CAR strategies include conditional activation CARs of heterodimers by dimerization of pairs of intracellular domains (see, eg, US Pat. No. 9,587,020), CARs. Split CAR, a homologous recombination of antigen binding, hinges, and internal domains (see, eg, US Patent Publication No. 2017/018347), two transmembrane domains linked to an antigen binding domain and a signaling domain, respectively. It has a multi-stranded CAR (see, eg, US Patent Publication No. 2014/0134142), a bispecific antigen-binding domain that allows non-shared links between them (see, eg, US Pat. No. 4,447,194). ), Or a CAR having a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, eg, US Pat. No. 8,409,577), or tandem CAR (eg, Hegde et al., J Clin Invest. 2016; 126 (8): 3036-3052); Inducible CAR (see, eg, U.S. Patent Publication Nos. 2016/0046700, 2016/0058857, 2017/01667877),. Also included are switchable CARs (see, eg, US Patent Publication No. 2014/0219975), and other designs known in the art.

Thus, provided herein includes inducible cells derived from genome-engineered iPSC differentiation, both iPSC and inducible cells include, but are not limited to, expression of exogenous hnCD16. Includes one or more CARs with modified modality. In one particular embodiment, the iPSC and its inducing cells comprise hnCD16, and a CAR that targets selected tumor or viral antigens, where the inducing cells are NK or T cells, and the inducing cells are: ADCC antibodies or duals that target different tumor antigens than those targeted by CAR in order to avoid or reduce tumor antigen escape while achieving dual targeting of the same tumor via hnCD16 binding. It can be used with one or more of the specificity, triple specificity, and multiple specificity antigens. In a further embodiment, the CAR-containing iPSC and its inducible T cells have a CAR inserted into the TCR constant region, resulting in TCR knockout and putting CAR expression under the control of the endogenous TCR promoter. In some embodiments, the engineered iPSC-derived derived TCR null CAR-T cells are CD16 (F176V and / or S197P) -natural or CD64-derived external domains, as well as natural or non-natural membranes. It further comprises hnCD16 having a penetrating, stimulating, and signaling domain. In another embodiment, the CAR-containing iPSC and its inducible NK cells insert the CAR into the NKG2A or NKG2D locus, resulting in NKG2A or NKG2D knockout and CAR expression under the control of the endogenous NKG2A or NKG2D promoter. Put.

3. 3. Extrinsic Introduced Cytokines and / or Cytokine Signaling Avoiding systemic high-dose administration of clinically relevant cytokines reduces the risk of dose-limiting toxicity from such actions and is cytokine-mediated cell autonomy. Gender is established. IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors to achieve lymphocyte autonomy without the need for additional administration of soluble cytokines. One or more partial or complete peptides of the cytokine are introduced into the cell to allow cytokine signaling with or without expression of the cytokine itself, thereby reducing the risk of cytokine toxicity. To maintain or improve cell proliferation, proliferation, proliferation, and / or effector function. In some embodiments, the introduced cytokines and / or their respective natural or modified receptors for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or transient.

FIG. 1 shows some configuration designs using IL15 as an example. Any transmembrane (TM) domain in the design of FIG. 1 may be native to the IL15 receptor or has been modified or replaced with a transmembrane domain of another membrane binding protein.

Design 1: IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics the trans presentation of IL15 without eliminating the cis presentation of IL15.

Design 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without excluding IL15 cis presentation and ensuring membrane binding of IL15. The recombinant protein has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 11. The recombinant protein comprises an IL15 propeptide downstream of the signal peptide. SEQ ID NO: 12 describes an exemplary DNA sequence encoding the amino acid sequence of SEQ ID NO: 11.
SEQ ID NO: 11
(414 amino acids)
SEQ ID NO: 12

(1242 Nucleic Acid)

Design 3: IL15Rα with a shortened intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking the trans presentation of IL15, maintaining membrane binding of IL15 and eliminating potential cis presentation. .. Such a construct comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 13, which is represented by SEQ ID NO: 14. It can be encoded by an exemplary nucleic acid sequence.
SEQ ID NO: 13
(Underlined 379 amino acids, signals and linker peptides)
SEQ ID NO: 14

(1140 nucleic acid)

Those skilled in the art will appreciate that the signal peptides and linker sequences described above are exemplary and will never limit their variations suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those of skill in the art. Those skilled in the art will appreciate that the signal peptide and / or linker sequence can be replaced with another sequence without altering the activity of the functional peptide that is derived or linked by the signal peptide.

The cytoplasmic domain of IL15Rα adversely affects the autonomic function of IL15-equipped effector cells in such a design, as the construct of Design 4: Design 3 has been shown to function in promoting the survival and proliferation of effector cells. Showing that it can be omitted without giving, Design 4 is a construct that provides another working alternative to Design 3, with essentially the entire IL15Rα removed, with the exception of the Sushi domain, IL15 at one end. And the other transmembrane domain fusion (mb-Sushi) and optionally include a linker between the Sushi domain and the transmembrane domain. The fused IL5 / mb-Sushi is expressed on the cell surface via the transmembrane domain of the membrane-binding protein. In constructs such as Design 4, unnecessary signal transduction via IL15Rα, including cis presentation, is eliminated if only the desired trans presentation of IL15 is retained. In some embodiments, the component comprising IL15 fused to the Sushi domain has an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 15. Included, which can be encoded by the exemplary nucleic acid sequence represented by SEQ ID NO: 16.
SEQ ID NO: 15
(Underlined 242 amino acids, signals and linker peptides)
SEQ ID NO: 16
(726 Nucleic Acid)

Those skilled in the art will appreciate that the signal peptides and linker sequences described above are exemplary and will never limit their variations suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those of skill in the art. Those skilled in the art will appreciate that the signal peptide and / or linker sequence can be replaced with another sequence without altering the activity of the functional peptide that is derived or linked by the signal peptide.

Design 5: Natural or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to allow constitutive signaling and maintain IL15 membrane binding and trans presentation.

Design 6: Natural or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling of cytokines and membrane-bound transpresentation. The common receptor γC is also called the common gamma chain or CD132 and is also known as the IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to many interleukin receptor receptor complexes including, but not limited to, IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

Design 7: Manipulated IL15Rβ, which forms a homodimer in the absence of IL15, is useful for producing constitutive signaling of cytokines.

In some embodiments, the cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18 and IL21, and / or one or more of their receptors are one of the designs of FIG. The above can be used to introduce iPSC and its inducing cells during iPSC differentiation. In some embodiments, cell surface expression and signal transduction of IL2 or IL15 is mediated by the construct set forth in any one of Designs 1-7. In some embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 is designed 5, 6, or 7 by using either a common receptor or a cytokine-specific receptor. Via the indicated construct. Any transmembrane (TM) domain in the design of FIG. 1 can be native to the receptor for each cytokine, or has been modified or replaced with a transmembrane domain of another membrane binding protein.

In iPSCs containing both CAR and exogenous cytokines and / or cytokine receptor signaling and inducible cells from them, CAR and IL are expressed in separate constructs or bicistronic constructs containing both CAR and IL. Co-expressed in. In some further embodiments, the form of IL15 represented by any of the construct designs in FIG. 1 comprises a self-cutting 2A coding sequence exemplified by, for example, CAR-2A-IL15 or IL15-2A-CAR. It can be linked to either the 5'or 3'end of the CAR expression construct via. Therefore, IL15 and CAR are in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises the IL15 of Design 3 of FIG. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises the IL15 of Design 3 of FIG. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises the IL15 of design 7 of FIG. When CAR-2A-IL15 or IL15-2A-CAR is expressed, the self-cleaving 2A peptide dissociates the expressed CAR and IL15, allowing the dissociated IL15 to be presented on the cell surface. Due to the bicistronic design of CAR-2A-IL15 or IL15-2A-CAR, both timing and quantity can be selected for incorporation, for example, inducible promoters for the expression of a single ORF. Allows coordinated expression of CAR and IL15 under the same regulatory mechanism. Self-cleaving peptides include foot-and-mouth disease virus (FMDV), horse rhinitis A virus (ERAV), Thosea asigna virus (TaV), and butatechovirus 1 (PTV-I) (Donnelly, ML, et al, J. Gen. Virus, 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virus., 72, 2727-2732 (2001)), as well as Tyrovirus (eg, Tyler mouse encephalomyelitis virus) and cerebral myocardium. It is found in members of the Picornavirus family, including the genus Cardiovirus, such as the flame virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to as "F2A", "E2A", "P2A", and "T2A", respectively.

The bicistronic CAR-2A-IL15 or IL15-2A-CAR embodiments disclosed herein for IL15 include any other cytokine provided herein, such as IL2, IL4, IL6, IL7. , IL9, IL10, IL11, IL12, IL18, and IL21 are also contemplated. In some embodiments, cell surface expression and signal transduction of IL2 is mediated by the constructs shown in any of Designs 1-7. In some other embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 uses either a common receptor and / or a cytokine-specific receptor, designed 5, 6, or. Via the construct shown in 7.

4. To avoid the problem of HLA-I- and HLA-II-deficiency allogeneic rejection, multiple HLA class I and class II proteins need to be matched for histocompatibility of allogeneic recipients. is there. Provided herein are iPSC cell lines in which expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced. HLA class I deficiency includes, but is limited to, functional deletion of any region of the HLA class I locus (chromosome 6p21), or beta2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. It can be achieved by deleting the HLA class I-related gene or reducing the expression level. For example, the B2M gene encodes a common subunit that is essential for cell surface expression of all HLA class I heterodimers. B2M null cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II related genes including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcription coactivator that functions through activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA null cells are HLA-II deficient. Provided here are iPSC lines in which both B2M and CIITA have been knocked out and their inducible cells, and the resulting inducible effector cells eliminate the need for MHC (major histocompatibility complex) matching. Allows allogeneic cell therapy by avoiding recognition and killing by host (allogeneic) T cells.

In some cell types, lack of class I expression causes lysis by NK cells. To overcome this "self-loss" response, HLA-G is knocked in as needed to avoid NK cell recognition and death of engineered iPSC-derived HLA-I and HLA-II-deficient effector cells. it can. In one embodiment, HLA-I and HLA-II deficient iPSCs and their inducible cells do not adversely affect the differentiation potential of iPSCs and the function of inducible effector cells, including inducible T cells and inducible NK cells, and hnCD16, and the need It further comprises one or both of CAR and IL depending on.

5. Genetically engineered iPSC strains and inducible cells provided herein In light of the above, the present application provides iPSC, iPS cell line cells, or inducible cells from them, which contain both hnCD16 and CAR expression. Here, the inducing cell is a functional effector cell obtained from the differentiation of iPSC including hnCD16 and CAR. In some embodiments, the inducing cells are hematopoietic cells, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic pluripotent progenitor cells. These include cells (MPPs), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. Not limited to. In some embodiments, functionally induced hematopoietic cells include effector cells such as T cells, NK cells, and regulatory cells.

In some embodiments, the inducing cells include NK cells or T cells. NK cells or T cells derived from iPSC containing both hnCD16 and CAR are observed with CAR-T alone treatment by combining the antibody with CAR targeting therapy if the antibody and CAR have specificity for different antigens of the tumor. It is useful for overcoming or alleviating tumor recurrence associated with tumor antigen escape. Induced CAR-T cells expressing hnCD16 have acquired ADCC and provide an additional mechanism for killing tumors in addition to CAR targeting. In some embodiments, the inducing cells include NK cells. NK cells derived from iPSCs containing hnCD16 and CAR have enhanced cytotoxicity and are effective in mobilizing bystander cells including T cells to infiltrate and kill tumor cells.

In addition, a polynucleotide encoding hnCD16 and a polynucleotide encoding CAR, and at least one exogenous cytokine and / or at least one exogenous cytokine that allows cytokine signaling that contributes to cell viability, sustained production, and / or proliferation. IPSC, iPS cell line cells, or derived cells from them, which contain a polynucleotide encoding a receptor (IL), are provided, and the iPSC line directs differentiation, viability, persistence, proliferation, and effector cells. It is possible to produce functionally induced cytokine cells with improved function. Exogenously introduced cytokine signaling includes signaling of any one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptide of the cytokine and / or its respective receptor for cytokine signaling is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or transient. In some embodiments, transient / transient expression of cell surface cytokines / cytokine receptors is mediated by RNA, including retroviruses, Sendaiviruses, adenoviruses, episomes, minicircles, or mRNAs. In some embodiments, exogenous cell surface cytokines and / or receptors contained in hnCD16 / CAR iPSC or its inducing cells allow IL7 signaling. In some embodiments, exogenous cell surface cytokines and / or receptors contained in hnCD16 / CAR ^- iPSC or its inducing cells allow IL10 signaling. In some embodiments, exogenous cell surface cytokines and / or receptors contained in hnCD16 / CAR iPSCs or their inducing cells allow IL15 signaling. In some embodiments of the hnCD16 / CAR / IL iPSC, IL15 expression is due to construct 3 in FIG. In some embodiments of the hnCD16 / CAR / IL iPSC, IL15 expression is due to construct 4 in FIG. The hnCD16 / CAR / IL iPSCs and their inducible cells of the above embodiments have cell proliferation, proliferation, proliferation, and / or effector function without contact with soluble cytokines additionally supplied in vitro or in vivo. Can be maintained or improved autonomously. In some embodiments, hnCD16 / CAR / IL iPSC and its inducible effector cells are used with antibodies to induce ADCC to target CAR by reducing or eliminating tumor antigen escape and subsequent tumor recurrence. It can be synergistic with tumor death.

IPSCs, iPS cell line cells containing HnCD16, CAR, IL, B2M knockouts and / or CIITA knockouts, and optionally HLA-G-encoding polynucleotides, or derived cells from them, are also provided to differentiate iPSCs. Can produce functional induced hematopoietic cells. In one embodiment of iPSC and its induced NK cells or induced T cells, the cells contain hnCD16 / CAR / IL / B2M ^{− / −} CIITA ^{− / −} and are deficient in both HLA-I and HLA-II. Can be used with antibodies that induce ADCC with CAR-targeted tumor cell killing, iPSC and its effector cells have improved persistence and / or viability. In some embodiments, effector cells have increased in vivo persistence and / or viability.

Thus, what is provided herein includes hnCD16 and CAR and, optionally, one, two, or all three of exogenous cytokines / receptors, B2M knockouts, and CIITA knockouts. Includes iPSCs from it, where when B2M is knocked out, polynucleotides encoding HLA-G are introduced as needed, and iPSCs direct differentiation to produce functional induced hematopoietic cells. be able to. The application also includes functional iPSC-induced hematopoiesis containing one, two, or all three of hnCD16 and CAR and, optionally, exogenous cytokines / receptors, B2M knockouts, and CIITA knockouts. Cells are also included, where when B2M is knocked out, a polynucleotide encoding HLA-G is introduced as needed, and the induced dendritic cells have definitive hematopoietic endothelial (HE) potential. Embryonic cells, definitive HE, CD34 hematopoietic cells, hematopoietic cell stem and progenitor cells, hematopoietic pluripotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T Includes, but is not limited to, cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

Therefore, the present application provides iPSCs containing any one of the following genotypes in Table 1 and functionally induced hematopoietic cells thereof. As provided, "IL" is IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which particular cytokine / receptor expression is selected. Represents one of them. If the iPSC and its functionally induced hematopoietic cells have a genotype containing both CAR and IL, CAR and IL are included in a bicistronic expression cassette containing the 2A sequence. In contrast, in some other embodiments, CAR and IL are in separate expression cassettes contained in iPSCs and their functionally induced hematopoietic cells. In one particular embodiment, the iPSC expressing both CAR and IL and its functionally induced effector cells contain IL15 in constructs 3 or 4 of FIG. 1, the IL15 construct with or with CAR. Separately included in the expression cassette.
Table 1: Applicable genotypes of the cells provided:

6. Further Modifications In some embodiments, the iPSC and its inducible effector cells containing any one of the genotypes in Table 1 are TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and It may further comprise a deletion or underexpression in at least one of the genes in the 6p21 region of the chromosome, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A _2A. Introduced or increased expression at least one of the R, TCR, Fc receptors, engagers, and surface-triggered receptors for binding to bispecific, multispecific or universal engagers. Can be further included.

A bispecific or multispecific engager is a fusion protein consisting of two or more single chain variable fragments (scFv) of different antibodies in which at least one scFv binds to an effector cell surface molecule and at least another one. One binds to tumor cells via tumor-specific surface molecules. Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition or coupling include CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, And, but not limited to, the chimeric Fc receptors disclosed herein. In some embodiments, CD16 expressed on the surface of effector cells for engagement recognition is described in Section I. CD16 (including F176V and optionally S197P) or CD64 extracellular domain, as described in 2, and hnCD16 containing natural or non-natural transmembrane, stimulating and / or signaling domains. In some embodiments, the CD16 expressed on the surface of effector cells for engagementer recognition is the hnCD16-based chimeric Fc receptor (CFCR). In some embodiments, the hnCD16-based CFcR comprises a transmembrane domain of NKG2D, a stimulation domain of 2B4, and a signaling domain of CD3ζ, and the extracellular domain of hnCD16 is the full length of the extracellular domain of CD64 or CD16 or Derived from the partial sequence, the extracellular domain of CD16 contains F176V and optionally S197P. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / Includes, but is not limited to, B, PSMA, PAMA, P-cadherin, ROR1. In one embodiment, the bispecific antibody is CD3-CD19. In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody is CD3-CD33. In yet another embodiment, the bispecific antibody is further modified IL15 as a linker between effector cells and tumor cell antigen binding domains, eg, effector NK cells that promote effector cell proliferation (some publications). The thing includes (called TriKE, or trispecific killer engager). In one embodiment, the TriKE is CD16-IL15-EPCAM or CD64-IL15-EPCAM. In another embodiment, the TriKE is CD16-IL15-CD33 or CD64-IL15-CD33. In yet another embodiment, the TriKE is NKG2C-IL15-CD33.

In some embodiments, the surface-triggered receptors of bispecific or multispecific conjugates can be endogenous to effector cells, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein are used, i.e., iPSCs containing the genotypes listed in Table 1 are further engineered to further manipulate T cells, NK cells, or One or more extrinsic surface-triggered receptors can be introduced into effector cells by directing the differentiation of iPSC into other effector cells containing the same genotype and surface-triggered receptor as the source iPSC.

7. Antibodies for Immunotherapy In some embodiments, in addition to the genome-engineered effector cells provided herein, an antibody or antibody fragment that targets an antigen associated with a condition, disease, or indicator is included. Additional therapeutic agents can be used in combination therapy with these effector cells. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, as an additional therapeutic agent for administered iPSC-derived effector cells, antibodies suitable for combination therapy include anti-CD20 (rituximab, bellzzumab, ofatumumab, ubrituximab, okaratuzumab, obinutuzumab), anti-HER2 (trastuzumab). , Parts Zumab), Anti-CD52 (Alemtuzumab), Anti-EGFR (Seltuximab), Anti-GD2 (Zinutuximab), Anti-PDL1 (Abelumab), Anti-CD38 (Daratumumab, Isatumumab, MOR202), Anti-CD123 (7G3, CSL362), Anti-SLAM7 ); And their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars, but not limited to them. In some embodiments, the iPSC-derived effector cells include hematopoietic lineage cells containing the genotypes listed in Table 1. In some embodiments, the iPSC-derived effector cells include NK cells containing the genotypes listed in Table 1. In some embodiments, the iPSC-derived effector cells include T cells containing the genotypes listed in Table 1. In some embodiments of compositions useful for the treatment of liquid or solid tumors, the composition comprises iPSC-derived NK or T cells, including hnCD16 and CAR, and antibodies with antigen specificity different from CAR. Including. In some further embodiments, the CAR contained in the hnCD16 expression-inducing NK or T cell targets any one of CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, and PDL1. , Cells can be used with any antibody that targets an antigen that is different from the antigen recognized by CAR to reduce or prevent tumor antigen escape from CAR targeting. For example, in one embodiment, if the CAR of an induced NK cell or T cell that also expresses hnCD16 targets CD123, the antibody used in combination with the cell is not an anti-CD123 antibody. In some other embodiments, iPSC-derived NK or T cells used in combination therapy include hnCD16, IL15, and CAR, where IL15 is co-expressed or separately expressed with CAR, IL15. Is any one of the forms shown in constructs 1-7 of FIG. 1, and the combination therapy comprises an antibody that targets a different antigen as compared to CAR. In some specific embodiments, IL15 is in the form of constructs 3, 4, or 7 if it is expressed with or separately from CAR.

8. Checkpoint Inhibitors Checkpoints are cell molecules, often cell surface molecules, that can suppress or downregulate the immune response when uninhibited. It has been shown that tumors select specific immune checkpoint pathways as a major mechanism of immune resistance, especially to tumor antigen-specific T cells. Checkpoint inhibitors (CIs) can block inhibitory checkpoints and restore immune system function by reducing the expression or gene products of checkpoint genes or reducing the activity of checkpoint molecules. It is an antagonist that can. The development of checkpoint inhibitors that target PD1 / PDL1 or CTLA4 has changed the outlook for oncology, and these agents have resulted in long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint block therapy, and recurrence remains a major concern. One aspect of the application provides a therapeutic approach for overcoming CI resistance by including genomically engineered functionally induced cells as provided in combination therapy with CI. In one embodiment of combination therapy, the inducing cells are NK cells. In another embodiment of combination therapy, the inducing cell is a T cell. In addition to exhibiting direct antitumor capacity, the induced NK cells provided herein resist PDL1-PD1-mediated inhibition, enhance T cell migration, and recruit T cells into the tumor microenvironment. It has been shown to have the ability to enhance T cell activation at the tumor site. Thus, tumor infiltration of T cells promoted by functionally potent genome-engineered induced NK cells causes the NK cells to synergize with T cell-targeted immunotherapy containing checkpoint inhibitors to suppress local immunosuppression. It shows that it can alleviate and reduce the amount of tumor.

In one embodiment, the induced NK cells for checkpoint inhibitor combination therapy are of hnCD16 and CAR, and optionally B2M knockout, CIITA knockout, and Gaia-negative cell surface cytokine and / or receptor expression. One, two, or three of the above, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally included. In some embodiments, the induced NK cell comprises any one of the genotypes listed in Table 1. In some embodiments, the inducible NK cells described above are deleted or deleted in at least one of the genes in the TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and chromosome 6p21 regions. further comprises or reduced expression, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, a 2A R, CAR, TCR, Fc receptors, engagers, and double It further comprises introduced or increased expression at least one of the surface-triggered receptors for specificity, multispecificity or binding to a universal engager.

In another embodiment, the induced T cells for checkpoint inhibitor combination therapy are hnCD16 and CAR, and optionally B2M knockout, CIITA knockout, and Gaia-negative cell surface cytokine and / or receptor expression. It comprises one, two, or three of them, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally included. In some embodiments, the induced T cell comprises any one of the genotypes listed in Table 1. In some embodiments, the aforementioned induced T cells are deleted or deleted in at least one of the genes in the TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and chromosome 6p21 regions. further comprises or reduced expression, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, a 2A R, CAR, TCR, Fc receptors, engagers, and double It further comprises introduced or increased expression at least one of the surface-triggered receptors for specificity, multispecificity or binding to a universal engager.

The above-mentioned induced NK cells or induced T cells are one, two or three of hnCD16 and CAR, and optionally B2M knockout, CIITA knockout, and exogenous cell surface cytokine and / or receptor expression. It is obtained by differentiating an iPSC clone strain containing, where, when B2M is knocked out, a polynucleotide encoding HLA-G is introduced as needed. In some embodiments, the iPSC clone strain described above is a deletion or deletion in at least one of the genes in the TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and chromosome 6p21 regions. further comprises or reduced expression, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, a 2A R, CAR, TCR, Fc receptors, engagers, and double It further comprises introduced or increased expression at least one of the surface-triggered receptors for specificity, multispecificity or binding to a universal engager.

Checkpoint inhibitors suitable for combination therapy with induced NK cells or induced T cells provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2). , TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctra4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA , CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B , NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (eg, 2DL1, 2DL2, 2DL3, 3DL1, 3DL2) antagonists. Includes, but is not limited to.

In some embodiments, the antagonist that inhibits any of the checkpoint molecules described above is an antibody. In some embodiments, the checkpoint inhibitory antibody is a mouse antibody, human antibody, humanized antibody, camel Ig, shark heavy chain only antibody (VNAR), Ig NAR, chimeric antibody, recombinant antibody, or antibody fragment thereof. Can be. Non-limiting examples of antibody fragments include Fab, Fab', F (ab) '2, F (ab) '3, Fv, single-stranded antigen-binding fragment (scFv), (scFv) 2, disulfide stabilization. Fv (dsFv), minibody, diabody, tribody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and others that maintain binding specificity of the entire antibody Antibody fragments are included, they can be more cost effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one or two or three or more checkpoint inhibitors are atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremerimmumab (anti-CTLA4). mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lilimumab (anti-KIR), monarizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), penbromab Anti-PD1 mAb), and derivatives thereof, functional equivalents, or at least one of their biocimirrors.

In some embodiments, antagonists that inhibit any of the checkpoint molecules described above are microRNA-based, as many miRNAs are found as regulators of immune checkpoint expression (Dragomir et al.,. Cancer Biol Med. 2018, 15 (2): 103-115). In some embodiments, the checkpoint antagonist miRNAs are miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197. , MiR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c, but not limited to these.

Some embodiments of combined therapy with induced NK cells or induced T cells provided include at least one checkpoint inhibitor that targets at least one checkpoint molecule, the inducible cells listed in Table 1. Has a genotype. Some other embodiments of the combined therapy with induced NK cells or induced T cells provided are two or three such that two, three or more checkpoint molecules are targeted. , Or more checkpoint inhibitors. In some embodiments of combination therapy comprising at least one checkpoint inhibitor and inducible cells having the genotypes listed in Table 1, the checkpoint inhibitor is an antibody, or humanized or Fc modified variant or fragment, or These functional equivalents or biosimilars, the checkpoint inhibitors, are produced by the inducing cells by expressing the exogenous polynucleotide sequence encoding the antibody, or fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the antibody, or fragment or variant thereof that inhibits the checkpoint, is bicistronic comprising both a separate construct or CAR and a sequence encoding the antibody or fragment thereof. Co-expressed with CAR in any of the constructs. In some further embodiments, the sequence encoding the antibody or fragment thereof is 5 of the CAR expression construct via a self-cleaving 2A coding sequence exemplified, such as CAR-2A-CI or CI-2A-CAR. It can be linked to either the'or 3'end. Therefore, the checkpoint inhibitor and CAR coding sequences are in a single open reading frame (ORF). When a checkpoint inhibitor is delivered and expressed and secreted as a payload by inducible effector cells capable of infiltrating the tumor microenvironment (TME), it counteracts the inhibitory checkpoint molecule when bound to TME, such as CAR. It activates effector cells by activating modality or activating receptor activation. In some embodiments, checkpoint inhibitors co-expressed with CAR are checkpoint molecules, PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, At least one of TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. Inhibits. In some embodiments, checkpoint inhibitors co-expressed with CAR in inducible cells having the genotypes listed in Table 1 are atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monarizumab. , Nivolumab, pembrolizumab, and their humanization, or Fc modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is atezolizumab, or a humanized version thereof, or an Fc modified variant, fragment or functional equivalent or biosimilar thereof. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is nivolumab, or a humanized version thereof, or an Fc modified variant, fragment or functional equivalent or biosimilar thereof. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is pembrolizumab, or a humanized version thereof, or an Fc modified variant, fragment or functional equivalent or biosimilar thereof.

In some other embodiments of combination therapy comprising at least one antibody that inhibits the inducer cell and checkpoint molecule provided herein, the antibody is not produced by or within the inducer cell. , Before, simultaneously with, or after administration of the induced cells having the genotypes listed in Table 1. In some embodiments, administration of one, two, three, or more checkpoint inhibitors in combination therapy with the provided induced NK cells or induced T cells is simultaneous or sequential. In one embodiment of combination therapy comprising inducible NK cells or inducible T cells with the genotypes listed in Table 1, the checkpoint inhibitors included in the treatment are atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43. , IPH33, limumab, monarizumab, nivolumab, pembrolizumab, and humanizations thereof, or Fc modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of combination therapy comprising inducible NK cells or inducible T cells with the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is atezolizumab, or a humanized or Fc modified variant thereof. Fragments and their functional equivalents or biosimilars. In some embodiments of combination therapy comprising induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is nivolumab, or a humanized or Fc modified variant thereof. Fragments and their functional equivalents or biosimilars. In some embodiments of combination therapy comprising induced NK cells or induced T cells with the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is pembrolizumab, or a humanized or Fc modified variant thereof. Fragments and their functional equivalents or biosimilars.

II. Methods of Targeted Genome Editing at Selected Loci of iPSC Genome editing, or genome editing, or gene editing, used interchangeably herein, involves inserting, deleting, and / or inserting DNA into the genome of a target cell. Or it is a kind of genetic engineering that is replaced. Targeted genome editing (compatible with "targeted genome editing" or "targeted gene editing") allows insertions, deletions, and substitutions at preselected sites within the genome. If an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or knocked down by the sequence deletion. Therefore, targeted editing can also be used to accurately disrupt endogenous gene expression. As used herein, the term "targeted integration" refers to a process involving the insertion of one or more exogenous sequences with or without deletion of the endogenous sequence at the insertion site. In contrast, randomly integrated genes are susceptible to positional effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomere regions are particularly susceptible to transgene silencing. Newly integrated genes can affect surrounding endogenous genes and chromatin, altering cell behavior and facilitating cell transformation. Therefore, inserting exogenous DNA into preselected loci such as the Safe Harbor locus or the Genome Safe Harbor (GSH) is for safety, efficiency, copy number control, and reliable gene response control. is important.

Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach. In a nuclease-independent targeted editing approach, homologous recombination is guided by a homologous sequence flanking the inserted exogenous polynucleotide via the host cell's enzymatic mechanism.

Alternatively, specific introduction of double-strand breaks (DSBs) with specific rare cut-end nucleases can result in more frequent targeted editing. Such nuclease-dependent targeted editing utilizes a DNA repair mechanism involving non-homologous end joining (NHEJ) that occurs in response to the DSB. In the absence of a donor vector containing an extrinsic genetic material, NHEJ often causes random insertion or deletion (indel) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing a pair of homology arms and flanking exogenous genetic material, the exogenous genetic material can be introduced into the genome during homologous recombination homologous directional repair (HDR), resulting in a "target". Incorporation is brought about.

Available endonucleases that can introduce specific targeted DSBs include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and RNA-induced CRISPR (clustered equidistant short-chain batch repeats) systems. Included, but not limited to. In addition, a DICE (dual integrase cassette exchange) system utilizing the fiC31 and Bxb1 integrase is also a promising tool for targeted integration.

ZFN is a targeted nuclease containing a nuclease fused to a zinc finger DNA binding domain. "Zinc finger DNA binding domain" or "ZFBD" means a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. Zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C ₂ H ₂ zinc fingers, C ₃ H zinc fingers, and C ₄ zinc fingers. A "designed" zinc finger domain is a non-naturally occurring domain whose design / configuration primarily processes information in a database that stores information on existing ZFP designs and join data, such as reasonable criteria. Due to the application of replacement rules and computerized algorithms for. See, for example, US Pat. Nos. 6,140,081, 6,453,242, and 6,534,261. See also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016494. A "selected" zinc finger domain is a non-naturally occurring domain whose production results primarily from empirical processes such as phage display, interaction traps, or hybrid selection. ZFNs are described in detail in US Pat. No. 7,888,121 and US Pat. No. 7,972,854, the full disclosure of which is incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of FokI nucleases with zinc finger DNA binding domains.

TALEN is a targeted nuclease containing a nuclease fused to the TAL effector DNA binding domain. "Transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" means the polypeptide domain of a TAL effector protein involved in the binding of a TAL effector protein to DNA. To do. The TAL effector protein is secreted by a plant pathogen of the genus Xanthomonas at the time of infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA-binding domains, and activate gene transcription at these sequences via the transactivation domain. The specificity of the TAL effector DNA binding domain depends on the effector variable number of incomplete 34 amino acid repeats containing a polymorphism at selected repeat positions called repetitive variable residues (RVDs). TALEN is described in more detail in US Patent Application 2011/01454940, which is incorporated herein by reference. The most recognized example of TALEN in the art is a fusion polypeptide of FokI nuclease to a TAL effector DNA binding domain.

Another example of the targeting nuclease used in the methods of the invention is fused to a targeted Spo11 nuclease, a DNA binding domain specific for the DNA sequence of interest, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, and the like. It is a polypeptide containing a Spo11 polypeptide having nuclease activity. See, for example, U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

Further examples of targeting nucleases suitable for the present invention, whether used individually or in combination, include Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1. Not limited to.

Other non-limiting examples of targeting nucleases are naturally occurring and from families including recombinant nucleases, cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr. CRISPR-related nucleases; restriction endonucleases; meganucleases; homing endonucleases and the like are included.

As an exemplary example, CRISPR / Cas9 requires two major components: (1) Cas9 endonuclease and (2) crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to the target DNA sequence containing the PAM and the seeding region proximal to the PAM. A chimeric guide RNA (gRNA) can be formed by combining crRNA and tracrRNA, and Cas9 can be directed to a selected sequence. These two components can be delivered to mammalian cells via transfection or transduction.

Insertion via DICE provides unidirectional integration of exogenous DNA that is tightly restricted to the small attB and attP recognition sites of each enzyme itself, using, for example, pairs of recombinases such as phiC31 and Bxb1. Since these targeted att sites are not naturally present in the mammalian genome, they must first be introduced into the genome of the desired integration site. See, for example, US Patent Application Publication No. 2015/0140665, the disclosure of which is incorporated herein by reference.

One aspect of the invention provides a construct containing one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific for the desired integration site, and the target integration method is to introduce the construct into a cell to perform site-specific homologous recombination by a cell host enzyme mechanism. Including making possible. In another embodiment, the method of targeted integration in a cell is to introduce a construct containing one or more exogenous polynucleotides into the cell and a ZFN expression cassette containing a DNA binding domain specific for the desired integration site. Is introduced into cells to allow insertion via ZFNs. In yet another embodiment, the method of targeted integration in the cell is to introduce into the cell a construct containing one or more exogenous polynucleotides and TALEN expression comprising a DNA binding domain specific for the desired integration site. Includes introducing a cassette into a cell to allow insertion via TALEN. In another embodiment, the method of targeted integration in the cell comprises introducing a construct containing one or more exogenous polynucleotides into the cell and a Cas9 expression cassette and a guide sequence specific for the desired integration site. Includes introducing gRNA into cells to allow insertion via Cas9. In yet another embodiment, the method of targeted integration in the cell is to introduce a construct containing one or more att sites of a pair of DICE recombinases into the desired integration site within the cell and one or more exogenous sites. It involves introducing a construct containing a polynucleotide into a cell and introducing an expression cassette for the DICE recombinase to allow targeted integration via DICE.

Promising sites for targeted integration are intra-gene or extra-gene regions of the human genome, theoretically predictable of newly integrated DNA without adversely affecting host cells or organisms. Includes, but is not limited to, the Safe Harbor locus, or Genome Safe Harbor (GSH), which can accommodate various expressions. A useful safe harbor needs to allow sufficient transgene expression to produce the desired level of vector-encoded protein or non-encoding RNA. Safe harbors must also not facilitate malignant transformation of cells or alter cell function. For a recombinant site to be a promising safe harbor locus, it must ideally meet, but is not limited to, regulatory elements or genes, as determined by sequence annotations: No disruption; an intergenic region within a dense region of genes, or a convergence position between two genes transcribed in opposite directions; a gene flanking a transcriptional activator encoded in a vector Keep distances to minimize the possibility of long-range interactions, especially with promoters of cancer-related genes and microRNA genes; a wide range of spatial and temporal expression sequence tags exhibiting ubiquitous transcriptional activity ( EST) Has apparently ubiquitous transcriptional activity, as reflected by the expression pattern. This latter feature is particularly important in stem cells, where chromatin remodeling usually results in silencing of some loci and potential activation of other loci during differentiation. Within a region suitable for extrinsic insertion, the exact locus selected for insertion lacks repeating elements and conserved sequences, and primers for amplification of the homology arm should be easily designed.

Sites suitable for human genome editing, or specifically targeted integration, include human orthologs of adeno-associated virus site 1 (AAVS1), chemokine (CC motif) receptor 5 (CCR5) locus, and mouse ROSA26 locus. Includes, but is not limited to. In addition, the human ortholog of the mouse H11 locus may also be a suitable site for insertion using the targeted integration compositions and methods disclosed herein. In addition, collagen and the HTRP locus can also be used as a safe harbor for targeted integration. However, validation of selected sites has been shown to be particularly necessary for stem cells for specific integration events, with insertion strategies such as promoter selection, exogenous gene sequence and placement, and construct design. Optimization is often needed.

Due to the targeted indel, the editing site is often contained in an endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene, including the targeted indel, is involved in the regulation and modification of the immune response. In some other embodiments, the endogenous gene, including the targeting indel, is a targeting modality, receptor, signaling molecule, transcription factor, drug target candidate, immune response regulation and regulation, or stem cell and / or progenitor cell. , And proteins derived from them that suppress cell engraftment, transport, homing, viability, self-renewal, persistence, and / or viability.

Thus, one aspect of the invention is a selected locus that includes a genomic safe harbor, or a continuous or transient gene such as B2M, TAP1, TAP2, or tapasin locus as provided herein. Provided are methods of targeted integration at preselected loci known or proven to be safe and well regulated for expression. In one embodiment, the genome safe harbor for the method of targeted integration is based on the criteria of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or genome safe harbor. Includes one or more desirable integration sites, including other loci that meet. In one embodiment, the method of targeted integration into a cell is to introduce into the cell a construct containing one or more exogenous polynucleotides and a pair and one or more homologous arms specific for the desired integration site. Desirable integration sites include AAVS1, CCR5, ROSA26, collagen, HTRP, including introducing into cells a construct containing multiple exogenous sequences to allow site-specific homologous recombination by cell-host enzymatic mechanisms. , H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of Genome Safe Harbor.

In other embodiments, the method of targeted integration into a cell is to introduce into the cell a construct containing one or more exogenous polynucleotides and to express a ZFN containing a DNA binding domain specific for the desired integration site. Desirable integration sites include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, including introducing cassettes into cells to allow insertion via ZFNs. Alternatively, it contains RUNX1 or another locus that meets the criteria of a genome safe harbor. In yet another embodiment, the method of targeted integration into a cell is to introduce into the cell a construct containing one or more exogenous polynucleotides and a TALEN comprising a DNA binding domain specific for the desired integration site. Desirable integration sites include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, including the introduction of an expression cassette into cells to allow insertion via TALEN. , Or other loci that meet the criteria of RUNX1, or Genome Safe Harbor. In another embodiment, the method of targeted integration in the cell comprises introducing a construct containing one or more exogenous polynucleotides into the cell and a Cas9 expression cassette and a guide sequence specific for the desired integration site. Desirable integration sites include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or, including the introduction of gRNA into cells to allow insertion via Cas9. Contains RUNX1 or other loci that meet the criteria of Genome Safe Harbor. In yet another embodiment, the method of targeted integration in a cell is to introduce a construct containing one or more att sites of a pair of DICE recombinases into the desired integration site within the cell and one or more exogenous sites. Desirable integration sites include AAVS1, CCR5, ROSA26, including introducing a construct containing a polynucleotide into the cell and introducing an expression cassette for the DICE recombinase to allow targeted integration via DICE. , Collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of Genome Safe Harbor.

In addition, as provided herein, the methods described above for targeted integration in safe harbors include polynucleotides of interest, such as safety switch proteins, targeted modalities, receptors, signaling molecules, transcriptions. Encodes factors, pharmaceutically active proteins and peptides, drug target candidates, and proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or viability of stem and / or progenitor cells. Used to insert a polypeptide. In some other embodiments, a construct containing one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the invention is a suicide gene encoding a safety switch protein. Suitable suicide gene systems for induced cell death include caspase-9 (or caspase 3 or 7) and AP1903, thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). Included, but not limited to. In addition, some suicide genotypes are cell type specific, for example, genetic modification of T lymphocytes by the B cell molecule CD20 can eliminate them upon administration of mAb rituximab. In addition, a modified EGFR containing an epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Thus, one aspect of the invention is the targeting of one or more suicide genes encoding a safety switch protein selected from caspase-9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20. Provides a built-in method.

In some embodiments, one or more exogenous polynucleotides incorporated by the methods herein are driven by an operably linked exogenous promoter contained in the construct for targeted integration. Promoters can be inducible or constitutive, and can be time-specific, tissue-specific or cell-type specific. Constitutive promoters suitable for the methods of the invention include cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C. (UBC) promoters are included, but not limited to these. In one embodiment, the exogenous promoter is CAG.

The exogenous polynucleotide incorporated by the methods herein can be driven by an endogenous promoter in the host genome at the site of integration. In one embodiment, the methods of the invention are used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the cellular genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the methods of the invention are used for targeted integration at the ROSA26 locus in the cell's genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the methods of the invention are used for targeted integration at the H11 locus in the cell's genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the methods of the invention are used for targeted integration at collagen loci in the cell's genome. In one embodiment, at least one integrated polynucleotide is driven by an endogenous collagen promoter. In yet another embodiment, the methods of the invention are used for targeted integration at the HTRP locus in the cell's genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. Theoretically, only the correct insertion at the desired location would allow gene expression of an exogenous gene driven by an endogenous promoter.

In some embodiments, one or more exogenous polynucleotides contained in the construct for the targeted integration method are driven by one promoter. In some embodiments, the construct comprises one or more linker sequences between two adjacent polynucleotides driven by the same promoter, enhancing physical separation between the moieties and maximizing access to the enzymatic mechanism. To do. The linker peptide of the linker sequence was selected to make the physical separation between the moieties (exogenous polynucleotide and / or the protein or peptide encoded from it) more flexible or tighter, depending on the associated function. Can consist of amino acids. The linker sequence can be cleaved by a protease or chemically, resulting in a separate moiety. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is either naturally produced by the host or introduced extrinsically. Alternatively, the cleavage site in the linker can be a site that can be cleaved upon exposure to selected chemicals such as cyanogen bromide, hydroxylamine, or low pH. Any linker sequence may serve a purpose other than providing a cleavage site. The linker sequence should allow for effective placement of the part with respect to another adjacent part for the part to function properly. The linker may also be a simple amino acid sequence long enough to prevent any steric hindrance between the moieties. Furthermore, the linker sequence can provide post-translational modifications including, but not limited to, for example, phosphorylation sites, biotination sites, sulfate sites, γ-carboxylation sites, and the like. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single undesired conformation. The linker may consist primarily of amino acids with small side chains such as glycine, alanine, and serine to provide flexibility. In some embodiments, about 80 or 90 percent or more of the linker sequence comprises glycine, alanine, or serine residues, in particular glycine and serine residues. In some embodiments, the G4S linker peptide separates the terminal processing and endonuclease domain of the fusion protein. In other embodiments, the 2A linker sequence allows two distinct proteins to be produced from a single translation. Suitable linker sequences can be easily identified empirically. In addition, the proper size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry site (IRES). In some embodiments, any two contiguous linker sequences are different.

A method of introducing a construct containing an exogenous polynucleotide for targeted integration into a cell can be achieved using a method of gene transfer into a cell known per se. In one embodiment, the construct comprises the backbone of a viral vector such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentiviral vector, a Sendai viral vector. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides to target cells such as target cells (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo). Will be done. In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides to target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic manipulation to introduce insertions, deletions, or substitutions via homologous recombination. Unlike the lentivirus, rAAV does not integrate into the host genome. In addition, the episome rAAV vector mediates a much higher rate of homologous directed gene targeting compared to transfection of conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the iPSC genome. In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein contain at least one genotype listed in Table 1.

III. Methods for Obtaining and Maintaining Genome-engineered iPSCs The present invention provides and targets methods for acquiring and maintaining genome-engineered iPSCs that include one or more targeted edits at one or more desired sites. Editing remains intact and functional at each selected editing site in the grown genome-engineered iPSC or iPSC-derived non-pluripotent cells. Targeted editing introduces insertions, deletions, and / or substitutions into the genome of iPSCs and cells derived from them, i.e., targeting integration and / or indels at selected sites. Many advantages of obtaining genome-engineered induced cells by editing and differentiating iPSCs as provided herein, as compared to direct manipulation of patient-derived peripheral blood-derived primary effector cells. Does include, but are not limited to: an unlimited source of designed effector cells; no need to repeatedly manipulate the effector cells, especially if multiple designed modalities are included; Rejuvenates due to extended telomeres and low wasting; the effector cell population is predominantly due to the ability to select mutants in the engineered iPSC provided herein. It is uniform in that there are no edit sites, number of copies, and allelic mutations, random mutations, or expression diversity.

In certain embodiments, genome-engineered iPSCs containing one or more targeted edits at one or more selected sites are long-term simple in cell culture media shown in Table 2 as Fate Maintenance Medium (FMM). Maintained, passaged, and proliferated as a single cell, where iPSCs retain targeted edits and functional alterations at selected sites. The components of the medium may be present in the medium in an amount within the optimum range shown in Table 2. FMM-cultured iPSCs have been shown to remain undifferentiated, maintain a basic or naive profile, and genomic stability that does not require culture washing or selection, and all three somatic lineages. , In vitro differentiation through embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation due to malformation easily occur. See, for example, U.S. Patent Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference.
Table 2: Illustrative media for reprogramming and maintenance of iPSCs

In some embodiments, genome-engineered iPSCs containing one or more targeted integration and / or indels are maintained, passaged, proliferated and TGFβ receptor in media containing MEKi, GSKi, and ROCKi. Free or essentially free of body / ALK5 inhibitors, where the iPSC retains intact and functional targeted edits at selected sites.

Another aspect of the present invention is to generate genome-manipulated non-pluripotent cells through targeted editing of iPSC or first by targeted editing, and then reprogram selected / isolated genome-manipulated non-pluripotent cells. To provide a method for generating genome-engineered iPSCs by obtaining iPSCs containing the same targeted edits as non-pluripotent cells. A further aspect of the invention provides genomically engineered non-pluripotent cells that are simultaneously being reprogrammed by introducing targeted integration and / or targeted indel into the cells, and the contacted non-pluripotent cells are reprogrammed. Under conditions sufficient for programming, reprogramming conditions include contacting non-pluripotent cells with one or more reprogramming factors and, if necessary, small molecules. In various embodiments of the method for simultaneous genomic manipulation and reprogramming, non-pluripotent cells are contacted with one or more reprogramming factors and, if necessary, small molecules before initiating reprogramming. , Or essentially simultaneously, can introduce targeted integration and / or targeted indels into non-target pluripotent cells.

In some embodiments, in order to simultaneously genome-manipulate and reprogram non-pluripotent cells, targeted integration and / or Indel also transforms non-pluripotent cells with one or more reprogramming factors and small molecules. Contact can be introduced into non-pluripotent cells after a multi-day process of reprogramming has begun, where the reprogramming cells include, but are not limited to, SSEA4, Tra181 and CD30. Before showing stable expression of the pluripotency gene, a construct-bearing vector is introduced.

In some embodiments, reprogramming reprograms non-pluripotent cells into at least one reprogramming factor and, optionally, TGFβ receptor / ALK inhibitor, MEK inhibitor, GSK3 inhibitor, and ROCK inhibitor. It is initiated by contact with the combination (FRM; Table 2). In some embodiments, the genome-engineered iPSC by any of the methods described above is further maintained and maintained using a mixture (FMM; Table 2) containing a combination of MEK inhibitor, GSK3 inhibitor, and ROCK inhibitor. Proliferate.

In some embodiments of the method of producing a genome-engineered iPSC, the method genome-manipulates the iPSC by introducing one or more targeted integrations and / or indels into the iPSC and at least listed in Table 1. It involves acquiring a genome-engineered iPSC having one genotype. Alternatively, methods of generating genome-engineered iPSCs are as follows: (a) Introduce one or more targeted edits into non-pluripotent cells to target genomes at selected sites and / or include indels. Obtaining engineered non-pluripotent cells and (b) one or more reprogramming factors for genome-manipulated non-pluripotent cells, and optionally TGFβ receptor / ALK inhibitors, MEK inhibitors, Contacting with a small molecule composition comprising a GSK3 inhibitor and / or a ROCK inhibitor to obtain a genome-engineered iPSC containing targeted integration and / or indel at a selected site. Alternatively, methods for generating genome-engineered iPSCs include (a) one or more reprogramming factors for non-pluripotent cells and, optionally, TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3. Initiate reprogramming of non-pluripotent cells by contacting with an inhibitor and / or a small molecule composition containing a ROCK inhibitor, and (b) genome one or more targeted integrations and / or indels. Reprogramming for Manipulation Includes introduction into non-pluripotent cells and (c) acquisition of cloned genome-engineered iPSCs containing targeted integration and / or indel at selected sites.

Reprogramming factors include OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136. Selected from the group consisting of TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1 and any combination thereof, their disclosures are incorporated herein by reference. One or more reprogramming factors can be in the form of polypeptides. Reprogramming factors can also be in the form of polynucleotides and are therefore introduced into non-pluripotent cells by vectors such as retroviruses, Sendaiviruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by episomal vectors. In various other embodiments, the one or more polynucleotides are introduced by the Sendai viral vector. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids. See, for example, U.S. Patent Application No. 62 / 571,105, the disclosure of which is incorporated herein by reference.

In some embodiments, the non-pluripotent cell is a plurality of constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted integration at the same or different selected sites. Introduced in. These exogenous polynucleotides engraft and transport suicide genes or targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or iPSCs or their inducing cells. , Homing, viability, self-renewal, persistence, and / or may include genes encoding proteins that promote viability. In some embodiments, the exogenous polynucleotide encodes an RNA including, but not limited to, siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides can be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue-specific or cell-type-specific promoters. Thus, polynucleotides can be expressed under conditions that activate the promoter, eg, in the presence of inducers, or in certain differentiated cell types. In some embodiments, the polynucleotide is expressed in cells differentiated from iPSCs and / or iPSCs. In one embodiment, the one or more suicide genes are capase-9 driven by a constitutive promoter, eg, CAG. These constructs, which contain different exogenous polynucleotides and / or different promoters, can be introduced into non-pluripotent cells simultaneously or sequentially. Non-pluripotent cells subjected to targeted integration of multiple constructs can be contacted with one or more reprogramming factors at the same time to initiate reprogramming at the same time as genome manipulation, which allows multiple cells in the same pool of cells. Genome-engineered iPSCs containing targeted integration of can be obtained. Thus, this robust method allows simultaneous reprogramming and manipulation strategies to induce cloned genome-manipulated hiPSCs with multiple modality integrated into one or more selected target sites. In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein contain at least one genotype listed in Table 1.

IV. A method for obtaining genetically engineered non-pluripotent cells by differentiating a genome-engineered iPSC A further aspect of the present invention provides a method for in vivo differentiation of a genome-engineered iPSC by dysplasia formation, the genome. Differentiated cells in vivo derived from engineered iPSCs retain intact and functional targeted editing, including targeted integration and / or indel, at the desired site. In some embodiments, differentiated cells in vivo derived from iPSC genome-engineered via teratomas are AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, Alternatively, it comprises one or more inducible suicide genes integrated into one or more desired sites, including RUNX1, or another locus that meets the criteria of Genome Safe Harbor. In some other embodiments, differentiated cells in vivo derived from iPSCs genome-engineered via malformations transport, homing, polynucleotides encoding targeting modalities, or stem and / or progenitor cells. Includes polynucleotides encoding proteins that promote viability, self-renewal, persistence, and / or viability. In some embodiments, in vivo-induced differentiated cells from iPSC genome-engineered via a teratoma containing one or more inducible suicide genes are endogenous genes involved in the regulation and mediation of the immune response. Further includes one or more indels of. In some embodiments, the indel is included in one or more endogenous checkpoint genes. In some embodiments, the indel is included in one or more endogenous T cell receptor genes. In some embodiments, the indel is included in one or more endogenous MHC class I suppressor genes. In some embodiments, the indel is included in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the indel is included in one or more endogenous genes including, but not limited to, the B2M, PD1, TAP1, TAP2, tapasin, TCR genes. In one embodiment, a genome-engineered iPSC containing one or more exogenous polynucleotides at selected sites further comprises targeted editing in a gene encoding B2M (beta-2-microglobulin).

In certain embodiments, genomically engineered iPSCs, including one or more genetically modified animals provided herein, are used and induced to induce hematopoietic cell lines or other specific cell types in vitro. The non-pluripotent cells that have been cultivated retain functional genetic modifications, including targeted editing, at selected sites. In one embodiment, the genome-engineered iPSC-derived cells are mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic pluripotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. Here, without limitation, these cells derived from genome-engineered iPSCs carry functional genetic modifications, including targeted edits at the desired site.

Applicable differentiation methods and compositions for obtaining hematopoietic cell lines derived from iPSCs include, for example, those described in International Application No. PCT / US2016 / 0444122, the disclosure of which is herein by reference. Be incorporated. As provided, methods and compositions for generating hematopoietic cell lines require scalable, monolayered EB formation under serum-free, feeder-free, and / or stroma-free conditions. In culture platforms that do not, via the definitive hematopoietic endothelium (HE) derived from pluripotent stem cells, including hiPSC. Cells that can differentiate according to the methods provided include a variety of cells that have transitioned directly from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, and to hematopoietic fate without going through pluripotent intermediates. It extends to cells of the lineage. Similarly, the cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to final differentiated cells and all intervening hematopoietic cell lines.

Methods of differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer cultures include contacting the pluripotent stem cells with BMP pathway activators and, optionally, bFGF. As provided, mesoderm cells derived from pluripotent stem cells are obtained and proliferated from the pluripotent stem cells without forming embryoid bodies. The mesoderm cells are then exposed to contact with BMP pathway activators, bFGF, and WNT pathway activators to create definitive hematopoietic endothelium (HE) potential without forming embryoid bodies from pluripotent stem cells. Obtain proliferating mesoderm cells with. Subsequent contact with bFGF and, optionally, with ROCK inhibitors and / or WNT pathway activators, mesodermal cells with definitive HE potential are similarly proliferated during differentiation. Differentiates into definitive HE cells.

The method for obtaining cells of the hematopoietic lineage provided herein is superior to EB-mediated pluripotent stem cell differentiation, which is uniform, with EB formation leading from moderate to minimal cell proliferation. This is because it does not allow monolayer culture, which is important for many applications that require proliferative growth, and uniform differentiation of cells within the population, resulting in labor and inefficiency.

The provided monolayer differentiation platform promotes differentiation into hematopoietic stem cells and definitive hematopoietic endothelials that lead to the induction of differentiated progeny such as T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale proliferation to enable the therapeutically appropriate number of pluripotent stem cell-derived hematopoietic cells to be delivered for a variety of therapeutic applications. In addition, monolayer cultures using the methods provided herein are functional hematopoietic strains that allow in vitro differentiation, ex vivo modification, and in vivo long-term hematopoiesis self-renewal, reconstruction and engraftment. Brings cells. As provided, iPSC-derived hematopoietic lineage cells include definitive hematopoietic endothelials, hematopoietic pluripotent progenitor cells, hematopoietic stem and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells. , NKT cells, B cells, macrophages, and neutrophils, but are not limited to these.

A method of directing the differentiation of pluripotent stem cells into cells of the definitive hematopoietic lineage, in which (i) pluripotent stem cells are contacted with a composition containing a BMP activator and optionally bFGF to pluripotency. Initiate differentiation and proliferation of endothelium cells from competent stem cells, and (ii) endothelium cells containing BMP activators, bFGF, and GSK3 inhibitors, optionally with TGFβ receptor / ALK inhibitors. Initiate differentiation and proliferation of endothelium cells with decisive HE potential from contact with a composition that does not contain, and (iii) ROCK inhibitors of endothelium cells with definitive HE potential; Contains one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, as well as Wnt pathway activators as needed, and TGFβ receptors as needed. / Includes contacting with an ALK inhibitor-free composition to initiate definitive hematopoietic endothelial differentiation and proliferation from pluripotent stem cell-derived mesodermal cells with definitive hematopoietic endothelial potential.

In some embodiments, the method involves contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor and not containing a TGFβ receptor / ALK inhibitor. It further comprises seeding and proliferating stem cells. In some embodiments, the pluripotent stem cell is an iPSC, or naive iPSC, or an iPSC containing one or more genetic imprints, from which one or more genetic imprints contained in the iPSC. It is retained by differentiated hematopoietic cells. In some embodiments of the method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the differentiation of pluripotent stem cells into cells of the hematopoietic lineage is monolayered with no embryonic body formation. It is a culture form.

In some embodiments of the methods described above, the definitive hematopoietic endothelial cell derived from the resulting pluripotent stem cell is CD34 +. In some embodiments, the definitive hematopoietic endothelial cells obtained are CD34 + CD43-. In some embodiments, the definitive hematopoietic endothelial cell is CD34 + CD43-CXCR4-CD73-. In some embodiments, the definitive hematopoietic endothelial cell is CD34 + CXCR4-CD73-. In some embodiments, the definitive hematopoietic endothelial cell is CD34 + CD43-CD93-. In some embodiments, the definitive hematopoietic endothelial cell is CD34 + CD93-.

In some embodiments of the methods described above, the method comprises (i) a definitive hematopoietic endothelial derived from pluripotent stem cells, a ROCK inhibitor; VEGF, bFGF, SCF, Flt3L, TPO, and IL7. Contact with one or more growth factors and cytokines selected from the group and, optionally, a composition containing BMP activator to initiate the differentiation of definitive hematopoietic endothelial into pre-T cell progenitor cells. And optionally (ii) pre-T cell progenitor cells, including one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but with VEGF, bFGF, TPO, BMP activation. Initiation of differentiation of pre-T cell progenitor cells into T cell progenitor cells or T cells by contact with a composition that does not contain one or more of the factors and ROCK inhibitors further comprises. In some embodiments of this method, the pluripotent stem cell-derived T cell progenitor cells are CD34 + CD45 + CD7 +. In some embodiments of this method, the pluripotent stem cell-derived T cell progenitor cell is CD45 + CD7 +.

In some further embodiments of the above-mentioned method for directing the differentiation of pluripotent stem cells into cells of hematopoietic lineage, this method (i) definitive hematopoietic endothelium derived from pluripotent stem cells. ROCK inhibitor; with a composition comprising one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally BMP activators. Contact to initiate definitive hematopoietic endothelial differentiation into pre-NK cell progenitor cells, and optionally (ii) pluripotent stem cell-derived pre-NK cell progenitor cells, SCF, Flt3L, IL3, Contact with a composition comprising one or more growth factors and cytokines selected from the group consisting of IL7, and IL15 and not containing one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors. It further comprises initiating differentiation of pre-NK cell progenitor cells into NK cell progenitor cells or NK cells. In some embodiments, the pluripotent stem cell-derived NK progenitor cell is CD3-CD45 + CD56 + CD7 +. In some embodiments, pluripotent stem cell-derived NK cells are CD3-CD45 + CD56 +, further defined by NKp46 +, CD57 + and CD16 + as needed.

Therefore, using the differentiation method described above, one or more populations of the following iPSC-derived hematopoietic cells can be obtained (i) iMPP-A, iTC-A2, iTC-B2, iNK-A2, And selected from CD34 + HE cells (iCD34), (ii) iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2 using one or more media selected from iNK-B2. One or more media selected from the definitive hematopoietic endothelium (iHE), (iii) iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2 using one or more media. One or more media selected from definitive HSCs, (iv) iMPP-A-based pluripotent progenitor cells (iMPP), (v) iTC-A2, and iTC-B2 were used. T cell progenitor cells (ipro-T), T cells using (vi) iTC-B2 (iTC), (vii) iNK-A2, and NK cells using one or more media selected from iNK-B2 Progenitor cells (ipro-NK) and / or (viii) NK cells (iNK), and iNK-B2. In some embodiments, the medium is:
a. iCD34-C contains one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally Wnt pathway activators. Includes and does not contain TGFβ receptor / ALK inhibitors.
b. iMPP-A is a BMP activator, ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. including.
c. iTC-A2 comprises one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, SCF, Flt3L, TPO, and IL7, and optionally BMP activators.
d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7.
e. iNK-A2 contains a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally BMP activators. ..
f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

In some embodiments, the genome-engineered iPSC-derived cells obtained from the methods described above are AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or genome. Includes one or more inducible suicide genes integrated into one or more desirable integration sites, including other loci that meet Safe Harbor criteria. In some other embodiments, the genome-engineered iPSC-derived cells are safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or Includes polypeptides encoding proteins that promote stem cell and / or progenitor cell transport, homing, viability, self-renewal, persistence, and / or viability. In some embodiments, genome-engineered iPSC-derived cells containing one or more suicide genes include, but are not limited to, checkpoint genes, endogenous T cell receptor genes, and MHC class I suppressor genes. It further comprises one or more indels contained in one or more endogenous genes involved in immune response regulation and mediation. In one embodiment, a genome-engineered iPSC-derived cell containing one or more suicide genes contains an indel in the B2M gene and the B2M is knocked out.

In addition, applicable dedifferentiation methods and compositions for obtaining second fate genome-manipulated hematopoietic cells from first-fate genome-manipulated hematopoietic cells are incorporated herein by reference, eg, the disclosure thereof. Includes those shown in International Publication No. WO2011 / 159726. The methods and compositions provided therein are capable of partially reprogramming initiated non-pluripotent cells into non-pluripotent intermediate cells by limiting the expression of the endogenous Nanog gene during reprogramming. It allows non-pluripotent intermediate cells to be subjected to conditions for differentiating the intermediate cells into the desired cell type. In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein contain at least one genotype listed in Table 1.

V. Therapeutic use of induced immune cells with functional modalities differentiated from genetically engineered iPSCs The present invention has been differentiated from genomically engineered iPSCs using the disclosed methods and compositions in some embodiments. A composition comprising an isolated population or subpopulation of functionally enhanced derived immune cells is provided. In some embodiments, the iPSC comprises one or more targeted gene edits that can be retained in iPSC-derived immune cells, and the genetically engineered iPSC and its inducible cells are suitable for cell-based adoption therapy. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived CD34 cells. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro T cells or T cells. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived immunomodulatory cells or bone marrow-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived genetically engineered immune cells are further modified ex vivo for improved therapeutic potential. In one embodiment, an isolated population or subpopulation of iPSC-derived genetically engineered immune cells comprises increasing the number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSC comprises increasing the number or proportion of type I NKT cells. In another embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSC comprises increasing the number or proportion of adaptive NK cells. In some embodiments, the isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, or bone marrow-derived suppressor cells derived from iPSC is allogeneic. In some other embodiments, the isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, or MDSCs derived from iPSCs is autologous.

In some embodiments, the iPSC for differentiation comprises a gene imprint selected to convey the desired therapeutic attributes in the effector cells, the cell developmental biology during differentiation is not disrupted, and the iPSC-derived differentiation described above. The gene imprint is retained and functional in hematopoietic cells.

In some embodiments, the genetic imprint of a pluripotent stem cell is (i) genome insertion, deletion, or deletion in the genome of the pluripotent cell during or after reprogramming the non-pluripotent cell to an iPSC. Includes one or more genetically modified modalities obtained by substitution, or (ii) one or more retainable therapeutic attributes of source-specific immune cells that are donor-specific, disease-specific, or therapeutic response-specific. The pluripotent cells are reprogrammed from source-specific immune cells, and the iPSC retains the therapeutic attributes of the therapeutic attribute source, which is also contained in iPSC-derived hematopoietic cells.

In some embodiments, the genetically modified modality is a safety switch protein, targeting modality, receptor, signaling molecule, transcription factor, pharmaceutically active protein and peptide, drug target candidate, or iPSC or inducible cell thereof. Includes one or more proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and regulation of immune responses, and / or viability. In some embodiments, the genetically modified iPSC and its inducible cells contain the genotypes listed in Table 1. In some other embodiments, the genetically modified iPSCs and their inducing cells containing the genotypes listed in Table 1 are: (1) TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5. , Or RFXAP, and one or more deletions or downregulation of any gene in the 6p21 region of the chromosome, and (2) HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1. Additional genetic modification modalities, including introduction or increased expression of A2AR, CAR, TCR, Fc receptors, or surface-triggered receptors for coupling with bispecific or multispecific or universal engagers. Including.

In yet some other embodiments, hematopoietic cells include the therapeutic attributes of source-specific immune cells for at least two combinations of: (i) expression of one or more antigen-targeted receptors,. (Ii) Modified HLA, (iii) Tumor resistance to microenvironment, (iv) Bystander immune cell recruitment and immunomodulation, (iv) Improved target specificity with reduced extratumor effects, (v) ) Improved homing, persistent, cytotoxic, or antigen escape rescue relief.

In some embodiments, iPSC-induced hematopoietic cells, including the genotypes listed in Table 1, and said cells are IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21. It expresses at least one cytokine and / or a receptor thereof, or any modified protein thereof, and at least CAR and hnCD16. In some embodiments, the engineered expression of cytokines and CARs is NK cell specific. In some other embodiments, the engineered expression of cytokines and CAR is T cell specific. In one embodiment, the CAR of the induced hematopoietic cell comprises a binding domain that recognizes any one of the CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, and PDL1 antigens. In some embodiments, antigen-specific induced effector cells target liquid tumors. In some embodiments, antigen-specific induced effector cells target solid tumors. In some embodiments, antigen-specific iPSC-induced effector cells can rescue tumor antigen escape.

By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases can be improved. In some embodiments, the iPSC-induced hematopoietic cells provided are for allogeneic adoptive cell therapy. In addition, the invention provides, in some embodiments, the therapeutic use of the therapeutic compositions described above by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject is an autoimmune disorder. Hematological malignancies, solid tumors, or infections associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

Examples of hematological malignancies include acute and chronic leukemia (acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple occurrences. Examples of solid cancers include, but are not limited to, myelogenous myelogenous tumors and myelogenous dysplasia syndromes: brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, Cancers of the bladder, kidney, head, neck, stomach, cervix, rectal, laryngeal, and esophagus include, but are not limited to. Examples of various autoimmune disorders include alopecia circumflexis, autoimmune hemolytic disease. Anemia, autoimmune hepatitis, dermatitis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerular nephritis, Graves' disease, Gillan Valley syndrome, idiopathic thrombocytopenic purpura, severe myasthenia , Several forms of myelogenous disease, polysclerosis, vesicles / varices, malignant anemia, nodular polyarteritis, polymyelitis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / Includes systemic sclerosis, Schegren's syndrome, systemic erythematosus, some forms of thyroiditis, some forms of vegetative inflammation, leukoplakia, granulomatosis with polyangiitis (Wegener's disease), but these Examples of viral infections include HIV- (human immunodeficiency virus), HSV- (simple herpesvirus), KSHV- (kaposi sarcoma-related herpesvirus), RSV- (respiratory folliculous virus), EBV. -(Epstein-Barvirus), CMV- (Cytomegalovirus), VZV (Vulgarite herpes zoster virus), Adenovirus-, Lentivirus-, BK polyomavirus-related diseases, but not limited to these.

Treatment using the induced hematopoietic lineage cells of the embodiments disclosed herein can be performed on a symptom-based basis or to prevent recurrence. Terms such as "treating" and "treatment" are used herein generally to mean obtaining the desired pharmacological and / or physiological effect. The effect can be prophylactic with respect to the complete or partial prevention of the disease and / or therapeutic with respect to the partial or complete cure of the disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers all interventions of a disease in a subject and includes: in a subject who is susceptible to the disease but has not yet been diagnosed with it. Preventing it from occurring, inhibiting the disease, that is, preventing its onset, or alleviating the disease, that is, retreating the disease. Therapeutic agents or compositions can be administered before, during or after the onset of the disease or injury. Treatment of ongoing diseases, where treatment stabilizes or alleviates unwanted clinical symptoms in the patient, is also of particular importance. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury that can be suppressed, ameliorated, and / or ameliorated by cell therapy at least one associated condition. In certain embodiments, the subject in need of cell therapy is a candidate for bone marrow or stem cell transplantation, a subject who has received chemotherapy or radiation therapy, a hyperproliferative disorder or cancer, such as a hematopoietic hyperproliferative disorder or cancer. Includes subjects who have or are at risk of having a tumor, such as those who have or are at risk of developing a solid tumor, who have or are at risk of having a viral infection or a disease associated with a viral infection. , It is intended not to be limited to these.

When assessing the responsiveness to treatments comprising the induced hematopoietic cells of the embodiments disclosed herein, the responses are clinical benefit rate, survival to death, pathological complete response, pathological response. Quantitative measurement, complete clinical remission, partial clinical remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulatory tumor cell depletion, circulatory marker response, and RECIST (response endpoint in solid tumors) It can be measured by at least one of the criteria.

Therapeutic compositions comprising induced hematopoietic lineage cells as disclosed can be administered to the subject before, during, and / or after other treatments. Thus, methods of combination therapy may include administration or preparation of iPSC-derived immune cells before, during, and / or after use of additional therapeutic agents. As mentioned above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components. Or a replacement factor thereof, including a vector, antibody, chemotherapeutic agent or radioactive moiety, or immunomodulator (IMiD) containing one or more polypeptide of interest. Administration of immune cells derived from iPSCs can be separated temporally, daily, or weekly from administration of additional therapeutic agents. Additional or alternative, administration can be combined with other bioactive agents or modality, such as, but not limited to, antitumor agents, non-drug therapies such as surgery.

In some embodiments of combinational cell therapy, the therapeutic combination comprises the iPSC-derived hematopoietic lineage cells provided herein and additional therapeutic agents that are antibodies or fragments thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, antibodies suitable for concomitant therapy as additional therapeutic agents against administered iPSC-derived hematopoietic cells include anti-CD20 (eg, rituximab, beltszumab, ofatumumab, ubrituximab, okaratsumab, obinutuzumab). However, it is not limited to these. Anti-HER2 (eg, trastuzumab, partszumab), anti-CD52 (eg, alemtuzumab), anti-EGFR (eg, seltuzumab), anti-GD2 (eg, ginutuximab), anti-PDL1 (eg, abelumab), anti-CD38 (eg, daratumumab, isatsuximab) , MOR202), anti-CD123 (eg, 7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc modified variants or fragments thereof or their functional equivalents or biosimilars.

In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. Checkpoints refer to cell molecules, often cell surface molecules, that can suppress or downregulate an immune response when uninhibited. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene products, or reduce the activity of checkpoint molecules. Checkpoint inhibitors suitable for combination therapy with induced effector cells, including NK cells or T cells, provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3. (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctra4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (eg, 2DL1, 2DL2, 2DL3, 3DL1, 3DL2). ), But is not limited to these.

Some embodiments of combination therapy comprising the provided inducible effector cells further comprise at least one inhibitor that targets the checkpoint molecule. Some other embodiments of combination therapy with the provided inducible effector cells include two, three, or more so that two, three, or more checkpoint molecules are targeted. Includes inhibitors of. In some embodiments, the effector cells for the combination therapies described herein are induced NK cells as provided. In some embodiments, the effector cells for combination therapy described herein are induced T cells. In some embodiments, the induced NK or T cells for combination therapy are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors can be administered in combination therapy at the same time, before, or after administration of the inducing effector cells. In some embodiments, the two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

In some embodiments, the antagonist that inhibits any of the checkpoint molecules described above is an antibody. In some embodiments, the checkpoint inhibitory antibody is a mouse antibody, human antibody, humanized antibody, camel Ig, shark heavy chain only antibody (VNAR), Ig NAR, chimeric antibody, recombinant antibody, or antibody fragment thereof. Can be. Non-limiting examples of antibody fragments include Fab, Fab', F (ab) '2, F (ab) '3, Fv, single-stranded antigen-binding fragment (scFv), (scFv) 2, disulfide stabilization. Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and others that maintain binding specificity of the entire antibody Antibody fragments are included, they can be more cost effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one or two or three or more checkpoint inhibitors are atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monarizumab, nivolumab, pembrolizumab, and theirs. Includes at least one of the derivatives or functional equivalents.

Combination therapy with inducible effector cells and one or more check inhibitors includes cutaneous T-cell lymphoma, non-Hodgkin lymphoma (NHL), mycobacterial sarcoma, Paget's reticular disease, Cesarly syndrome, granulomatous flaccid skin, lymphoma-like Scarlet disease, chronic lichenoid scab, acute psoriasis lichenoid lymphoma, CD30 + cutaneous T-cell lymphoma, secondary skin CD30 + large cell lymphoma, non-bacterial sarcoma CD30 skin large T-cell lymphoma, polymorphic T-cell lymphoma, Renato lymphoma, subcutaneous T-cell lymphoma, vascular central lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, Hodgkin lymphoma (HL), head and neck tumor, squamous cell carcinoma, rhombic myoma, Lewis lung cancer ( LLC), non-small cell lung cancer, esophageal squamous epithelial cancer, esophageal adenocarcinoma, renal cell cancer (RCC), colon cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine cancer (SCNC) ), Liver cancer, lymphoma, liver cancer, oral squamous epithelial cancer, pancreatic cancer, papillary thyroid cancer, intrahepatic bile duct cell cancer, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal cancer. It can be applied to the treatment of liquid cancer and solid cancer.

In some embodiments other than inducible effector cells as provided herein, the combination for therapeutic use comprises one or more additional therapeutic agents, including chemotherapeutic agents or radioactive moieties. Chemotherapeutic agents are found to be cytotoxic antitumor agents, that is, to preferentially kill tumor cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells. Refers to chemical agents that are used therapeutically to prevent or reduce the growth of neoplastic cells. Chemotherapeutic agents are also sometimes referred to as antitumor or cytotoxic drugs or agents and are well known in the art.

In some embodiments, the chemotherapeutic agent is an anthracycline, alkylating agent, alkyl sulfonate, aziridine, ethyleneimine, methylmelamine, nitrogen mustard, nitrosourea, antibiotics, antimetabolites, folic acid analogs, purines. Includes analogs, pyrimidine analogs, enzymes, podophylrotoxins, platinum-containing agents, interferons, and interleukins. Exemplary chemotherapeutic agents include alkylating agents (cyclophosphamide, mechloretamine, mefarin, chlorambusyl, hair methylmelamine, thiotepa, busulfan, carmustin, romustine, semstin), metabolic antagonists (methotrexate, fluorouracil, floxuridine). , Citarabin, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, bindesin), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxanthrone) , Vincristine, actinomycin D, prikamycin, puromycin, and gramicidin D), paclitaxel, corhitin, cytocarasin B, etoposide, maytancin, and amsacrine, but not limited to these. Additional agents include amineglutetimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, romustin (CCNU), carmustin (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L- Asparaginase, azacitidine, bebacizumab, bexaloten, bleomycin, voltezomib, busulfan, carsterone, capecitabin, selecoxib, setuximab, cladribine, cloflavin, citarabin, dacarbazine, deniroykin diftitox, diethylstilbestrol, docetaylvestrol Ramstin, etopocid, ethinylestradiol, exemestane, floxuridine, 5-fluorouracil, fludarabin, flutamide, fulvestrant, gefitinib, gemcitabine, goseleline, hydroxyurea, ibritsumomab, idarubicin, ifosphamide, imatinib, interferon alpha 2 , Irinotecan, letrozole, leucovorin, leuprolide, levamizole, mechloretamine, megestrol, melphaline, mercaptopurine, methotrexate, methoxalen, mitomycin C, mitotan, mitoxanthrone, nandrolone, nofetumomab, oxaliplatin, paclitamoxifen, paclitamoxifen , Pegaspargase, pentostatin, pipobroman, plicamycin, polyfeprosan, porphimer, procarbazine, quinacrine, rituximab, salgramostim, streptozocin, tamoxifen, temozolomide, teniposide, testlactone, thioguanine, thiotepa, topetecanthre , Trethinoin, uracil mustard, balrubicin, binorelbin, and zoredronate. Other suitable agents are those approved for use in humans, including those approved as chemotherapeutic or radiotherapeutic agents and those known in the art. Such agents are available in many standard physician and oncologist references (eg, Goodman & Gilman's The Pharmaceutical Bases of Therapeutics, Ninth Edition, McGraw-Hill Education, NY, 1995) or NY, 1995. You can refer to any of the websites (fda.gov/cder/cancer/druglistfrarne.hm), both of which will be updated from time to time.

Immunomodulators (IMiD) such as thalidomide, lenalidomide, and pomalidomide stimulate both NK and T cells. As provided herein, IMiD can be used with iPSC-derived therapeutic immune cells for the treatment of cancer.

In addition to the isolated population of iPSC-derived hematopoietic cells contained in the therapeutic composition, compositions suitable for administration to a patient include one or more pharmaceutically acceptable carriers (additives) and / or dilutions. It may further comprise an agent (eg, a pharmaceutically acceptable medium, eg, a cell culture medium), or other pharmaceutically acceptable component. The pharmaceutically acceptable carrier and / or diluent is determined in part by the particular composition administered and by the particular method used to administer the therapeutic composition. Therefore, there is a wide variety of suitable formulations of the therapeutic composition of the present invention (e.g., the disclosure of incorporated by reference in its entirety herein, by reference Remington's Pharmaceutical ^Sciences, 17 th ed.1985 I want).

In one embodiment, the therapeutic composition comprises T cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises NK cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises CD34 + HE cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises a pluripotent cell-derived HSC made by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises a pluripotent cell-derived MDSC made by the methods and compositions disclosed herein. Therapeutic compositions comprising a population of iPSC-derived hematopoietic lineages disclosed herein are administered individually by intravenous, intraperitoneal, enteric, or tracheal administration methods, or in combination with other suitable compounds. It is possible to influence the desired therapeutic target.

These pharmaceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition at about 3 to about 10. As such, the buffer can be as high as about 5% by weight of the total composition. Electrolytes such as, but not limited to, sodium chloride and potassium chloride may also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH of one of the pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

The invention also provides, in part, the use of pharmaceutically acceptable cell culture media in the particular compositions and / or cultures of the invention. Such compositions are suitable for administration to human subjects. Generally speaking, any medium that supports the maintenance, proliferation, and / or health of iPSC-derived immune cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is serum-free and / or feeder-free medium. In various embodiments, the serum-free medium is free of animal components and can optionally be protein-free. If desired, the medium may contain a pharmaceutically acceptable recombinant protein. Medium without animal components refers to media in which the components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free medium, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, protein-free medium is defined as substantially protein-free. Those skilled in the art will never limit the formulation of media suitable for use in the present invention by way of example of the media described above, and there are many suitable media known to those skilled in the art and available. Will understand.

Hematopoietic lineage cells derived from isolated pluripotent stem cells are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, pros. It can have T cells, pro-NK cells, CD34 + HE cells, HSCs, B cells, bone marrow-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic lineage cells are from about 95% to about 100% T cells, NK cells, pro T cells, pro NK cells, CD34 + HE cells, or bone marrow. It has suppressor cells (MDSC). In some embodiments, the invention is about 95% T cells, NK cells, pro T cells, pro NK cells, CD34 + HE cells, or bone marrow-derived suppressor cells for treating a subject in need of cell therapy. Provided are therapeutic compositions with purified T cells or NK cells, such as compositions with an isolated population of (MDSC).

In one embodiment, the combined cell therapy comprises a population of NK cells derived from a genomically engineered iPSC comprising a Therapeutic antibody or fragment thereof, and the genotypes listed in Table 1, wherein the induced NK cells. , HnCD16 and CAR. In another embodiment, combination cell therapy comprises a population of T cells derived from a genomically engineered iPSC comprising a Therapeutic antibody or fragment thereof, and the genotypes listed in Table 1, wherein the induced T cells. Includes hnCD16 and CAR. In some embodiments, the combination cell therapies include rituximab, bertuzumab, ofatumumab, ubrituximab, okaratuzumab, obinutzumab, trastuzumab, pertuzumab, alemtuzumab, cerutuximab, dinutuximab, seltuximab, dinutuximab, seltuximab, dinutuximab, abelumab, daratsumab Includes at least one of them, as well as a population of NK or T cells derived from genomically engineered iPSCs containing the genotypes listed in Table 1, wherein the induced NK or T cells include hnCD16 and CAR. .. In yet some other embodiments, the combination cell therapy comprises a population of NK cells or T cells derived from erotuzumab and a genomically engineered iPSC containing the genotypes listed in Table 1, where induction. NK or T cells include hnCD16 and CARs that target CD19, BCMA, CD38, CD20, CD22, or CD123. In some additional embodiments, the combination cell therapy comprises a population of NK cells or T cells derived from rituximab and a genomically engineered iPSC containing the genotypes listed in Table 1 and are derived NK cells or T cells contain hnCD16 and CAR as well as one or more exogenous cytokines.

As will be appreciated by those skilled in the art, both autologous and allogeneic hematopoietic cells derived from iPSCs based on the methods and compositions herein can be used in cell therapies as described above. In the case of autologous transplantation, the isolated population of induced hematopoietic lineage cells is HLA-matched in whole or in part with the patient. In another embodiment, the inducible hematopoietic lineage cell does not match the subject and HLA, where the inducible hematopoietic lineage cell is an NK cell or T cell having HLA I null and HLA II null.

In some embodiments, the number of induced hematopoietic cells in the therapeutic composition is at least 0.1 × 10 ⁵ cells, at least 1 × 10 ⁵ cells, at least 5 × 10 ⁵ cells, at least 1 × 10 per dose. ⁶ cells, at least 5 × 10 ⁶ cells, at least 1 × 10 ⁷ cells, at least 5 × 10 ⁷ cells, at least 1 × 10 ⁸ cells, at least 5 × 10 ⁸ cells, at least 1 × 10 ⁹ cells, or at least 5 × 10, ⁹ cells. In some embodiments, the number of induced hematopoietic cells in the therapeutic composition is from about 0.1 × 10 ⁵ cells to about 1 × 10 ⁶ cells per dose, from about 0.5 × 10 ⁶ cells per dose to about. 1 × 10 ⁷ cells, about 0.5 × 10 ⁷ cells to about 1 × 10 ⁸ cells per dose, about 0.5 × 10 ⁸ cells to about 1 × 10 ⁹ cells per dose, about 1 × 10 ⁹ cells per dose ~ About 5 × 10 ⁹ cells, about 0.5 × 10 ⁹ cells per dose to about 8 × 10 ⁹ cells, about 3 × 10 ⁹ cells per dose to about 3 × 10 ¹⁰ cells, or any range in between. .. Generally, when the patient's 60 kg, 1 × 10 ⁸ cells / dose is converted to 1.67 × 10 ⁶ cells / kg.

In one embodiment, the number of induced hematopoietic lineage cells in the therapeutic composition, or the number of partial or single immune cells in the umbilical cord blood, or at least 0.1 × 10 ⁵ cells / kg body weight, At least 0.5 × 10 ⁵ cells / kg body weight, at least 1 × 10 ⁵ cells / kg body weight, at least 5 × 10 ⁵ cells / kg body weight, at least 10 × 10 ⁵ cells / kg body weight, at least 0.75 × 10 ⁶ cells / Body kg body weight, at least 1.25 × 10 ⁶ cells / kg body weight, at least 1.5 × 10 ⁶ cells / kg body weight, at least 1.75 × 10 ⁶ cells / kg body weight, at least 2 × 10 ⁶ cells / kg body weight , At least 2.5 × 10 ⁶ cells / kg body weight, at least 3 × 10 ⁶ cells / kg body weight, at least 4 × 10 ⁶ cells / kg body weight, at least 5 × 10 ⁶ cells / kg body weight, at least 10 × 10 ⁶ cells / kg body weight kg body weight, at least 15 × 10 ⁶ cells / kg body weight, at least 20 × 10 ⁶ cells / kg body weight, at least 25 × 10 ⁶ cells / kg body weight, at least 30 × 10 ⁶ cells / kg body weight, 1 × 10 ⁸ cells / kg Body weight, 5 × 10 ⁸ cells / kg body weight, or 1 × 10 ⁹ cells / kg body weight.

In one embodiment, a dose of induced hematopoietic lineage cells is delivered to the subject. In one exemplary embodiment, the effective amount of cells provided to the subject is at least 2 × 10 ⁶ cells / kg, at least 3 × 10 ⁶ cells / kg, at least 4 × 10 ⁶ cells / kg, at least 5 × 10. ⁶ cells / kg, at least 6 × 10 ⁶ cells / kg, at least 7 × 10 ⁶ cells / kg, at least 8 × 10 ⁶ cells / kg, at least 9 × 10 ⁶ cells / kg, or at least 10 × 10 ⁶ cells / kg , Or more cells / kg, including all intervening cell doses.

In another exemplary embodiment, the effective amount of cells provided to the subject is about 2 × 10 ⁶ cells / kg, about 3 × 10 ⁶ cells / kg, about 4 × 10 ⁶ cells / kg, about 5 ×. 10 ⁶ cells / kg, about 6 × 10 ⁶ cells / kg, about 7 × 10 ⁶ cells / kg, about 8 × 10 ⁶ cells / kg, about 9 × 10 ⁶ cells / kg, or about 10 × 10 ⁶ cells / kg kg or more cells / kg, including all intervening cell doses.

In another exemplary embodiment, the effective amount of cells to be provided to a subject is about 2 × 10 ⁶ cells / kg to about 10 × 10 ⁶ cells / kg, from about 3 × 10 ⁶ cells / kg to about 10 × 10 ⁶ cells / kg, about 4 × 10 ⁶ cells / kg to about 10 × 10 ⁶ cells / kg, about 5 × 10 ⁶ cells / kg to about 10 × 10 ⁶ cells / kg, 2 × 10 ⁶ cells / kg to about 6 × 10 ⁶ cells / kg, 2 × 10 ⁶ cells / kg to about 7 × 10 ⁶ cells / kg, 2 × 10 ⁶ cells / kg to about 8 × 10 ⁶ cells / kg, 3 × 10 ⁶ cells / kg to about 6 × 10 ⁶ cells / kg, 3 × 10 ⁶ cells / kg to about 7 × 10 ⁶ cells / kg, 3 × 10 ⁶ cells / kg to about 8 × 10 ⁶ cells / kg, 4 × 10 ⁶ cells / kg to about 6 × 10 ⁶ cells / kg, 4 × 10 ⁶ cells / kg to about 7 × 10 ⁶ cells / kg, 4 × 10 ⁶ cells / kg to about 8 × 10 ⁶ cells / kg, 5 × 10 ⁶ cells / kg to about 6 × 10 ⁶ cells / kg, 5 × 10 ⁶ cells / kg to about 7 × 10 ⁶ cells / kg, 5 × 10 ⁶ cells / kg to about 8 × 10 ⁶ cells / kg or 6 × 10, ⁶ cells / kg to about 8 × 10 ⁶ cells / kg, including all intervening cell doses.

In some embodiments, the therapeutic use of induced hematopoietic lineage cells is a single dose therapy. In some embodiments, the therapeutic use of induced hematopoietic lineage cells is multiple dose therapy. In some embodiments, the multi-dose treatment is daily, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days. , Once every 45 days, every 50 days, or any number of times in any number of days in between.

Compositions comprising a population of induced hematopoietic lineage cells of the present invention can be sterile, suitable for administration to human patients, and can be administered immediately (ie, can be administered without further treatment). Immediately administered cell-based compositions mean that the composition does not require any further treatment or manipulation prior to transplantation or administration to the subject. In other embodiments, the invention provides an isolated population of induced hematopoietic lineage cells that are proliferated and / or modified prior to administration of one or more agents. For induced hematopoietic lineage cells genetically engineered to express recombinant TCR or CAR, the cells are activated and proliferated using, for example, the method described in US Pat. No. 6,352,694. be able to.

In certain embodiments, the primary and co-stimulating signals for induced hematopoietic lineage cells can be provided by different protocols. For example, the agent providing each signal can be in solution or bound to a surface. When bound to a surface, the agent can be bound to the same surface (ie, in "cis" formation) or to a separate surface (ie, in "trans" formation). Alternatively, one drug can be attached to the surface and the other drug can be present in solution. In one embodiment, the agent providing the co-stimulation signal can bind to the cell surface and the agent providing the primary activation signal is in solution or attached to the surface. In certain embodiments, both agents can be in solution. In another embodiment, the agent can be in soluble form, followed by cells or antibodies expressing Fc receptors, or artificially intended for use in the activation and proliferation of T lymphocytes in embodiments of the invention. It can be cross-linked to the surface of other binding agents that bind to agents as disclosed in US Patent Application Publication Nos. 2004/0101519 and 2006/0034810 for antigen-presenting cells (aAPC).

Some variations in dosage, frequency, and protocol will inevitably occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose, frequency and protocol for the individual subject.

The following examples are provided for illustration purposes only and are not for limitation purposes.

Example 1-Materials and Methods Single cell passages and high throughput 96 to effectively select suicide systems under the control of various promoters and test in combination with various safeharbor locus integration strategies. The applicant's proprietary hiPSC platform, which enables well plate-based flow cytometry sorting, was used to allow the derivation of cloned hiPSCs by single or multiple genetic modifications.

Maintenance of hiPSCs in small molecule cultures: hiPSCs were routinely passaged as single cells when the culture confluency reached 75% -90%. In the case of single cell dissociation, hiPSC was washed once with PBS (Mediatic), treated with Accutase (Millipore) at 37 ° C. for 3 to 5 minutes, and then pipetted to ensure single cell dissociation. The single cell suspension was then mixed in equal volumes with conventional medium, centrifuged at 225 xg for 4 minutes, resuspended in FMM and seeded on a matrigel-coated surface. Subcultures were typically 1: 6 to 1: 8, transferred to tissue culture plates pre-coated with Matrigel at 37 ° C. for 2-4 hours, and FMM was fed every 2-3 days. Cell cultures were maintained in a humidified incubator set at 37 ° C. and 5% CO2.

Using CRISPR: ROSA26 targeted insertion for ZFN manipulation of human iPSCs, targeted editing of the desired modality as an example, in genome editing via ZFN, 2 million iPSCs are 2.5 μg of ZFN- It was transfected with L (FTV893), a 2.5 μg mixture of ZFN-R (FTV894) and a 5 μg donor construct for AAVS1 targeted insertion. For genome editing via CRISPR, 2 million iPSCs were transfected with a mixture of 5 μg ROSA26-gRNA / Cas9 (FTV922) and a 5 μg donor construct for ROSA26 targeted insertion. Transfections were performed using a Neon transfection system (Life Technologies) using parameters 1500 V, 10 ms, 3 pulses. On the second or third day after transfection, if the plasmid contained an artificial promoter-driver GFP and / or RFP expression cassette, flow cytometry was used to measure transfection efficiency. On the 4th day after transfection, puromycin was added to the medium at a concentration of 0.1 μg / ml for the first 7 days and at a concentration of 0.2 μg / ml 7 days later to select target cells. During the selection of puromycin, cells were passaged into fresh Matrigel-coated wells on day 10. After day 16 of puromycin selection, viable cells were analyzed by flow cytometry for the percentage of GFP + iPS cells.

Bulk Sorting and Clone Sorting of Genome-Edited iPSCs: iPSCs, including genome-targeted editing using ZFNs or CRISPR-Cas9, were bulk-sorted and clone-sorted GFP + SSEA4 + TRA181 + iPSC 20 days after puromycin selection. Single-cell dissociation-targeted iPSC pools were freshly prepared for optimal performance in Hanks balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1 x penicillin / streptomycin (Mediatech), and 10 mM It was resuspended in a cold staining buffer containing Hepes (Mediatic). Conjugate primary antibodies such as SSEA4-PE, TRA181-Alexa Fluor-647 (BD Biosciences) were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used in 7 μL in 100 μL of staining buffer per million cells. The solution was washed once with stain buffer, spun down at 225 g for 4 minutes, resuspended in stain buffer containing 10 μM thiazobib and maintained on ice for sorting by flow cytometry. Sorting by flow cytometry was performed by FACS Maria II (BD Biosciences). In bulk sorting, GFP + SSEA4 + TRA181 + cells were gated and sorted into 15 ml standard tubes filled with 7 ml FMM. In clone sorting, 100 μM nozzles were used to drain the sorted cells directly into a 96-well plate at a concentration of 3 events per well. Each well was pre-filled with 200 μL of FMM supplemented with 5 μg / mL fibronectin and 1 × penicillin / streptomycin (Mediatech) and pre-coated overnight with 5 × matrigel. For 5x Matrigel precoating, 1 aliquot of Matrigel is added to 5 mL DMEM / F12, incubated overnight at 4 ° C. for proper resuspension, and finally added to a 96-well plate at 50 μL per well, 37. Incubate overnight at ° C. 5 × Matrigel is aspirated just prior to adding medium to each well. When sorting was complete, the 96-well plate was centrifuged at 225 g for 1-2 minutes before incubation. The plate was allowed to stand for 7 days. On day 7, 150 μL of medium was removed from each well and replaced with 100 μL of FMM. Wells resupplied an additional 100 μL of FMM 10 days after sorting. Colonization was detected as early as day 2, and most colonies grew within 7-10 days after sorting. In the first passage, wells were washed with PBS and dissociated with 30 μL of accutase at 37 ° C. for about 10 minutes. The need for long-term accutase treatment reflects the compactness of colonies that did not play in long-term culture. After the cells are found to be dissociated, 200 μL of FMM is added to each well and pipetted several times to destroy the colonies. The dissociated colonies are transferred to another well of a 96-well plate pre-coated with 5x Matrigel and centrifuged at 225 g for 2 minutes prior to incubation. This one-to-one passage is done to spread the early colonies before growth. Subsequent passages are routine with acutase treatment for 3-5 minutes and 1: 4–1: 8 growth in 75-90% confluency into large wells pre-coated with 1 × matrigel of FMM. Was done in. Each cloned cell line was analyzed for GFP fluorescence level and TRA1-81 expression level. Nearly 100% GFP + and TRA1-81 + clone strains were selected for further PCR screening and analysis. Flow cytometric analysis was performed on the Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC).

Example 2-Construction and Design of Cell Surface Expressed Cytokines for Autonomous Induced Cells In this application, substitution of exogenous soluble recombinant cytokines not only supports in vitro induction of hematopoietic cells from iPSCs. It has also been shown to support in vivo persistence and survival of induced effector cells. Avoiding systemic high-dose administration of clinically relevant cytokines reduces the risk of dose-limiting toxicity from such actions and establishes cytokine-mediated cell autonomy. FIG. 1 shows some configuration designs using IL15 as an example. In particular, in Design 3, IL15Rα, which has a shortened intracellular domain, is fused to IL15 at the C-terminus via a linker, mimicking the trans presentation of IL15, maintaining membrane binding of IL15, and potential cis presentation. Demonstrate to eliminate. As an alternative to Design 3, Design 4 essentially removes the entire IL15Rα, but at one end fuses with the IL15 and at the other with the transmembrane domain (mb-Sushi), and optionally the Suchi domain and membrane. The exception is the Sushi domain, which has a linker between the transmembrane domains. The fused IL5 / mb-Sushi is expressed on the cell surface via the transmembrane domain of the membrane-binding protein. In constructs such as Design 4, unnecessary signal transduction via IL15Rα, including cis presentation, is eliminated if only the desired trans presentation of IL15 is retained.

Example 3-Step-by-step manipulation of iPSC and verification of modification-induced NK cells Induced pluripotent stem cells (iPSCs) express high-affinity non-cleavable CD16 (hnCD16) and lose HLA-I by knockout of B2M gene. , Loss of HLA-II by knockout of CIITA, overexpression of nonclassical HLA molecule HLA-G, and expression of linked IL15 / IL15Rα constructs were continuously engineered. After each step of operation and before the next step of operation, the iPSC was sorted for the desired phenotype. Manipulated iPSCs can be maintained in vitro or differentiated into NK cells after about 44 days of differentiation and proliferation to give about 1E6 mature NK cells from a single iPSC input. These induced NK cells can be cryopreserved and delivered to patients on demand.

In this exemplary figure, iPSCs genetically engineered to include hnCD16 expression have enhanced cytotoxicity. CD16 levels of iNK from unmanipulated iPSC are very low, and the endogenous CD16 receptor expressed by NK cells from peripheral blood is cleaved from the cell surface following activation of NK cells. In contrast, non-cleavable versions of iNKs derived from iPSCs engineered to contain hnCD16 express modified CD16 and maintain constant expression levels by avoiding shedding of CD16. To do. In induced NK cells, non-cleavage CD16 increases the expression of TNFα and CD107a, which are indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC). ADCC is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells.

Unlike NK cells, mature T cells from the primary (ie, natural / natural sources such as peripheral blood, cord blood, or other donor tissue) do not express CD16. In the present invention, a function that allows iPSC containing the expressed exogenous non-cleavable CD16 not only to express exogenous CD16 but also to perform a function through the gained ADCC mechanism without impairing the developmental biology of T cells. It was unexpected that it could differentiate into a typical T cell.

To achieve complex modifications of HLA, hnCD16 iPSC strains are gRNA pairs that target B2M with a plasmid expressing Cas9 nickase engineered to provide less off-target effects compared to wild-type Cas9. Transfected. B2M ^{− / −} and HLA I-deficient clones using targeted editing were further analyzed by clone genomic DNA sequencing and confirmed to have small deletions or insertions resulting in a B2M knockout phenotype. B2M knockout creates HLA I-deficient cells.

The hnCD16 expression and HLA class I deficient iPSC were then genetically engineered to introduce the HLA-G / B2M fusion protein using, for example, a lentivirus. A modified version of HLA-G avoids cleavage and further enhances the persistence of HLA class I modified iPSCs. The resulting iPSC strain was then genetically engineered to contain the IL15 / IL15Rα fusion protein. Various modified iPSC strains (hnCD16; hnCD16 / B2M ^{− / −} , hnCD16 / B2M ^{− / −} CIITA ^{− / −} ; hnCD16 / B2M ^{− / −} HLA-G; hnCD16 / B2M ^{− / −} CIITA ^{− / −} HLA− As G; hnCD16 / B2M ^{− / −} HLA-G / IL15-IL15Rα; hnCD16 / B2M ^{− / −} CIITA ^{− / −} HLA-G / IL15-IL15Rα), the modified IPSCs that maintain their hematopoietic differentiation potential They differentiated into NK cells or T cells, respectively, via CD34 + cells with decisive HE potential.

Flow cytometry of the obtained mature iPSC-derived NK cells in FIG. 2 shows stepwise manipulation of hnCD16 expression, B2M knockout, HLA-G expression, and IL15 / IL15Rα expression, and expression of treatment-related proteins by iPSC genome manipulation. It is shown that the changes are maintained during hematopoietic differentiation without disrupting the development of directed cells to the desired cell fate in vitro.

Example 4-IPSC-derived natural killer (NK) cell telomere shortening with improved function and persistence occurs with cell aging and is associated with stem cell dysfunction and cell aging. Here, it is shown that mature iNK cells obtained through directed differentiation of iPSCs contain telomeres that are longer than adult peripheral blood NK cells. Telomere length was determined using the 1301 T-cell leukemia strain as a control (100%) adjusted for the DNA index of _{G0 / 1} cells by flow cytometry of iPSC, adult peripheral blood NK cells, and iPSC-derived NK cells. Was done. As shown in FIG. 3, iPSC-derived NK cells maintain telomere length significantly longer than adult peripheral blood NK cells (p = .105, ANOVA), and iPSC-derived NK cells. Shows high likelihood of proliferation, viability, and persistence.

Mature, differently engineered iPSC-derived NK cell lines were incubated with allogeneic CD8 + T cells primed for the same iNK donor at various E: T (effector: target) ratios, respectively. The results are the average of two donors and only for each target iNK cell genotype is normalized to% of the target cells. After 48 hours, the remaining iNK target cells were counted by flow cytometry. As shown in FIG. 4, B2M ^{− / −} / HLA-I deficiency caused iNK cell loss due to T cell recognition and cytotoxicity, while B2M knockout caused allogeneic CD8 + T cells. Eliminates in vitro recognition of iNK cells manipulated by.

B2M ^{− / −} iPSC has improved persistence due to its ability to avoid T cell-mediated killing. However, these cells may be exposed to peripheral NK-induced recognition and killing. When differently engineered iPSC strains were incubated with allogeneic PBMCs, the Incubite Zoom imaging system was used to measure the respective loss of iPSC over time. As shown in FIG. 5A, loss of HLA-I (B2M ^{− / −} ) on iPSC results in increased cytotoxicity and loss of iPSC compared to hnCD16 iPSC. However, as shown in FIG. 5B, expression of HLA-G on B2M ^{− / −} iPSC can at least partially reverse the loss of HLA-I deficient cells. Therefore, expression of HLA-G rescues B2M ^{− / −} iPSC from killing by allogeneic NK cells.

To test cell function in vivo, SKOV-3-luciferase ovarian tumor cells were transplanted into NSG mice by intraperitoneal injection (IP) and then on day 4 alone or 1E7 hnCD16 / B2M ^{− / −} /. Treated by a single dose of anti-HER2 antibody in combination with HLA-G iNK cells. Tumor progression was measured by IVIS (in vivo imaging system) imaging to monitor tumor progression. As shown in the IVIS image of each mouse in FIG. 6A or the time course of tumor progression by IVIS imaging in FIG. 6B, a single dose of hnCD16 / B2M ^{− / −} HLA-GINK is used in an in vivo xenograft model of ovarian cancer. , Induced tumor regression.

In addition, expression of exogenous IL15 / IL15Rα in iPSCs and iNKs indicates directed differentiation of iPSCs into induced NK cells in vitro and survival of induced iNK cells, independent of the addition of soluble exogenous IL15. Was shown to promote. As shown in FIG. 7A, hnCD16 iPSC, hnCD16 / B2M ^{− / −} HLA-G iPSC differentiates better when soluble IL15 is added to the medium, while expressing hnCD16 / B2M ^{− / −} HLA-G iPSC. IL15 / IL15Rα differentiated equally well with or without soluble IL15. The effect of exogenous IL15 / IL15Rα expression on induced iNK cells was demonstrated in FIG. 7B, which illustrates the soluble IL15 titration test. As shown in FIG. 7B, iNK cells were thoroughly washed and returned to culture for 7 days at soluble IL15 concentrations in the range of 0 ng / ml to 10 ng / ml, but with proliferation of iNK cells expressing IL15 / IL15Rα. Growth was shown to be independent of soluble IL15 in culture.

Expression of the IL15 / IL15Rα construct enhances the persistence of iNK in vivo in the absence of soluble IL15. 8 million hnCD16 / B2M ^{− / −} / HLA-G or hnCD16 / B2M ^{− / −} / HLA-G / IL15 iNK cells are immunodeficient NOG mice (FIG. 8A) or NOG transgenic mice expressing human IL15 (FIG. 8A). The adopted child was introduced to 8B). As shown in FIG. 8A, only cells expressing the IL15 / IL15Rα construct persisted in vivo in the absence of soluble IL15, as well as both iNK genotypes in the presence of serum IL15 (FIG. 8B). .. Therefore, expression of IL15 (IL15-IL15Rα) linked to the IL15 receptor alpha chain not only promotes NK cell-oriented genome-engineered iPSC differentiation, but also the absence of exogenous growth factors in vitro and in vivo. Supports the survival of engineered induced NK cells below.

The co-expression of CAR and the IL15 / IL15Rα construct was then investigated for the potential synergistic effect between the two modalities in creating a highly effective, persistent and targeted NK cell therapy. NK-CAR is a chimeric antigen receptor optimized for NK cells to mediate a potent increase in NK cell signaling, eg, CD16, NKp44, NKp46, NKG2D, 2B4 (CD244), CD137 (41BB). ), IL21, DAP10, DAP12, and / or CD3ζ, which comprises at least one domain (transmembrane domain, costimulatory domain, or cytoplasmic signaling domain) derived from a molecule expressed in NK cells. The NK cell-optimized CAR (NK-CAR) used in the demonstration herein has a backbone containing an NKG2D transmembrane domain, a 2B4 co-stimulation domain, and a CD3ζ signaling domain containing one or more ITAMs. As shown in FIG. 22, about 250 U / ml exogenous human IL-2 NK-CAR19 expression-inducing NK cells with or without co-expression of IL15 / IL15Rα construct (IL-15RF in FIG. 22). Was cultured for 9 days in the presence and absence of. Therefore, expression of IL15 / IL15Rα eliminated the soluble cytokine dependence of NK-CAR19 iNK cells. Expression of IL15 / IL15Ra also promotes in vitro persistence as well as in vivo iNK persistence (FIG. 31A) and enhances antigen-driven proliferation in vivo (FIG. 31B). The alternative IL15 construct was also used to test the ability of induced cells to support survival, persistence, and proliferation, including co-expression of CAR and IL in bicistronic vectors.

Next, in the Nalm6 and Raji xenograft models, mice treated with CAR-IL15 / IL15Ra iNK cells show extended survival compared to the control group. A total of 2.5 × 10 ⁵ Razi-luciferase (Razi-luc) cells were IV injected into immunodeficient NSG mice. The next day, mice were treated with 5 × 10 ⁶ unmodified iNK cells, CAR iNK cells, or CAR-IL15 / IL15Ra iNK cells. Tumor progression was monitored by weekly bioluminescence imaging (BLI) (not shown), overall survival was monitored, and is shown in FIG. 32A. As shown, CAR-IL15 / IL15Ra iNK was statistically superior to CAR iNK in prolonging survival in this highly aggressive dissemination model of B-cell lymphoma (p = 0.018). , CAR-IL15 / IL15Ra iNK vs. CAR iNK). In addition, in the leukemia model, Nalm6 cells were implanted intraperitoneally in NSG mice. Mice 4 days later, IL15 / IL15RA or without, were treated with 1 × 10 ⁷ of iNK cells expressing either conventional 1928Z CAR construct or NK centric CAR. Tumor progression in individual mice was measured by bioluminescence imaging (not shown) and overall survival of each treatment group was shown and compared in FIG. 32B, where CAR-IL15 / IL15Ra iNK prevented tumor progression and animals. It was a cell that could maintain the survival of the mouse.

Thus, these induced NK cells with modified HLA class I and / or II expressing hnCD16 and / or IL15 have increased resistance to immune detection and / or prolong in vivo persistence. Therefore, it provides a universal source of off-the-shelf treatment regimens for solid tumors as well as hematological malignancies. In addition, iPSC-NK cells also induce T cell migration from the circulatory system in vivo, as shown by the T cell recruitment assay described in more detail in Examples 7 and 8.

Examples 5-CAR and inducible NK cells expressing hnCD16 synergize with antibodies in elimination of target cells Derivative NK cells from CAR-expressing iPSCs are obtained according to the indicated differentiation platform described herein. It was. FIG. 9 shows a CD56 + NK cell population, PBNK (peripheral blood NK) cells, iPSC-NK cells, T CAR-iPSC-NK cells (inducible NK cells that express CAR designed for T cells), and CAR4 (meso). Expression of surface markers of CD56 and CD3, transcriptional marker GFP, and NK cell surface receptors NKp44, CD16, at the gates of -iPSC-NK cells (inducible NK cells expressing CAR specifically designed for NK cells), The phenotype of CAR-expressing iPSC-derived NK cells using FasL flow cytometry analysis is shown. In this example, the exemplary T CAR has the structure of SS1-CD28-41BBζ, while the NK CAR has the structure of SS1-NKG2D-2B4ζ (CAR4), both targeting mesocellin-expressing tumor cells. Induced NK cells expressing both T-CAR and NK-CAR may lose KIR (CD158a, h, b1 / b2, e1 / e2, and I; not shown) expression compared to PBNK cells. found.

CAR4 expression-inducing NK when co-cultured with europium-loaded meso-high target cells such as K562meso cells (Fig. 10A) and A1847 cells (Fig. 10B) at various effector-to-target ratios (E: T). The antitumor activity of the cells was demonstrated. Mean ± SD of% specific tumor cell lysis is shown and the data represent three europium release assays. As shown in FIG. 10, iPSC-NK cells, T-CAR (meso) -iPSC-NK cells, and CAR4 (-)-iPSC-NK cells (CAR4 without scFV) are NK-CAR4 (meso) -iPSC. -The ability to kill both meso ^high targets was low compared to NK cells. Clone-derived CAR4 (meso) -iPSC-NK cells (Nos. 1 and 4) reproduce potent cell lytic activity as pooled (non-cloned) CAR4 (meso) -iPSC-NK against meso ^high targets.

In order to evaluate the activity of CAR4-iPSC-NK cells in vivo, killing of A1847 ovarian cancer cells was tested in a mouse xenograft model. In FIG. 11, PBNK cells, iPSC-NK cells, T CAR (meso) -iPSC-NK cells, and CAR4 (meso) -iPSC-NK cells were compared. Luciferase-expressing A1847 cells were inoculated into the abdominal cavity of NSG mice, and 4 days later, each NK cell of 1.5E7 was injected into the abdominal cavity once. In this assay, IL15 was administered to promote the survival and proliferation of NK in vivo, but as shown in Example 6, exemplary IL15 for imparting autonomy of induced NK cells in vivo. This procedure can be omitted or replaced by expressing the / IL15Rα construct. Mice were monitored and tumor volume was quantified by bioluminescence imaging (BLI) up to day 49. As shown in FIG. 11, treatment with NK cells resulted in a significant reduction in tumor mass after 7 days (P <0.0001) compared to untreated tumor-bearing mice, CAR4- after 28 days. Maximum antitumor activity was found in iPSC-NK cells (P = 0.0044 compared to iPSC-NK cells; P <0.0001 compared to T CAR-iPSC-NK cells). This significantly improved the antitumor activity of CAR4-iPSC-NK cells compared to PB-NK cells, iPSC-NK cells, and T-CAR-iPSC-NK cells and persisted at all time points ( FIG. 12A), iPSC-NK cells (P = 0.0017, HR = 0.2236, 95% CI: 0.009016-0.2229) and T-CAR-iPSC-NK cells (P = 0.0018, HR). = 0.2153, 95% CI: 0.009771-0.2511), and compared to PB-NK cells (P = 0.0018, FIG. 12B), the viability was significantly improved. Blood, spleen, and ascitic fluid were tested for the presence of NK cells during the course of treatment to assess the in vivo persistence of NK cells after injection. On day 10, CAR4-iPSC-NK cells were compared to PB-NK cells, iPSC-NK cells, circulatory system (FIG. 13A, average 4.28%, P = 0.0143), spleen (FIG. 13B). , Average 7.22%, P = 0.0066), and ascitic fluid (FIG. 13C, average 9.80%, P = 0.0410). After 21 days, the number of CAR4-iPSC-NK cells returned to the same level as PB-NK cells and iPSC-NK cells. By day 28, few NK cells are found in these tissues. In summary, these studies show that NK-CAR-expressing iPSC-derived NK cells have improved antitumor activity compared to CAR-non-expressing PB-NK or iPSC-NK cells, and T-CAR-expressing iPSC- Demonstrate that it mediates improved activity compared to NK cells.

Further in view of Example 4 above, the aggregated data presented herein express both NK CAR and hnCD16 in addition to B2M ^{− / −} , HLA-G, and IL15 / IL15Rα. Supports enhanced inducible NK cell products. FIG. 30 shows the maturation of NK cells, as shown by increased production of Granzyme B associated with NK killing ability, and increased expression of KIR2DL3 and KIR2DL1 that grant NK cell licensing status to acquire effector function. Is enhanced in hnCD16-CAR-IL-15 / IL-15Ra iNK cells. The in vitro cytotoxicity of hnCD16-CAR-IL15 / IL15Ra iNK cells against Nalm6 and ARH-77 target cell lines was investigated. FIG. 33A compares the cytotoxicity of hnCD16-CAR-IL15 / IL15Ra iNK cells at increasing E: T ratios in a 4-hour cytotoxicity assay against Nalm6 / CD19 + and Nalm6 / CD19-cells, as described above. Shows CD19-specific killing by cells. In addition, ARH-77 / CD19 + leukemic cells (FIG. 33B) or ARH-77 / CD19- cells (FIG. 33C) were used to modify the direct cytotoxicity and rituximab-induced ADCC of hnCD16-CAR-IL15 / IL15Ra iNK cells. INK cells were used as controls and measured using a 4-hour cellular cytotoxicity assay with or without rituximab. The results showed that hnC16-CAR-IL15 / IL15Ra iNK cells mediate both CAR induction and ADCC for B cell malignancies in vitro. In further functional analysis, parental ARH-77 cells (CD19 +) and ARH-77 CD19-cells were transduced with a red fluorescent tag and a green fluorescent tag, respectively. These cells were then mixed 1: 1 and used as target cells in a long-term cytotoxicity assay utilizing various iNK cell populations as effector cells in the presence or absence of rituximab antibody. The frequency of green CD19- and red CD19 + targets was measured throughout the assay using the Incucite ™ imaging system to quantify cytotoxicity to both target cells within a single well. .. In the mixed culture cytotoxicity assay shown in FIG. 34, data show remaining targets for both target types (CD19 + or CD19-) normalized to controls for effector-free cells (tumor cells alone). Plotted as cell frequency, effector cell hnCD16, CAR, and IL15 / IL15Ra modalities exert a synergistic effect in eradicating both CD19 + and CD19- targets in mixed culture cytotoxicity assays in vitro. It is shown to do.

The induced NK cells provided herein provide a consistent and universal therapeutic allogeneic cell product that is in vivo, persistent and has improved killing, eg, incorporating therapeutic antibodies. When used in combination therapy with ADCC, or with T cell-targeted immunotherapy by incorporating a checkpoint inhibitor as further described in Example 7, synergistic effects are achieved.

Example 6-Enhanced induced T cell clone hiPSC for combination therapy to rescue CAR antigen escape is transduced with a high affinity non-cleavable CD16 Fc receptor (hnCD16) herein. Differentiated into induced T cells according to the indicated differentiation protocol provided. FIG. 14A shows the expression of hnCD16 on the cell surface of the hiPSC, as seen by flow cytometry. FIG. 14B shows the expression of hnCD16 on the cell surface of hiPSC-derived CD8ab + T cells.

Induced T cells expressing hnCD16 are co-cultured with tumor target cells in the presence and absence of antibodies in increasing effector-target ratios and the cytotoxicity of the cells is assessed by fluorescence live cell imaging of the target cells. did. FIG. 15 shows that hnCD16-expressing iPSC-derived T cells have acquired ADCC's ability for antibody-labeled target cells.

Next, as shown in FIG. 16, the cloned hiPSC was transduced with a lentivirus containing a chimeric antigen receptor (CAR) and an hnCD16 Fc receptor to express both CAR and hnCD16 on the surface of the hiPSC. To show that expression of CAR and hnCD16 does not disrupt hematopoietic differentiation, CAR + hnCD16 + hiPSC was differentiated using the provided iCD34 differentiation platform. Expression of both CAR and hnCD16 in the CD34 + population after 10 days of differentiation has been shown by flow cytometry. The CAR + hnCD16 + iCD34 cells were then differentiated into NK or T cells and the expression of CAR and hnCD16 in their respective induction effector cells was monitored by flow cytometry. Expression of CAR and hnCD16 is maintained on the cell surface of both hiPSC-derived NK and T cells.

CAR-hnCD16 iT cells perform hnCD16-mediated ADCC in addition to CAR-mediated cytotoxicity to achieve double cytotoxic targeting of tumor cells targeted by CD19-CAR-T cells. To investigate whether to alleviate CAR antigen escape, tumor cells expressing CD19 and CD20 (CD19 + / +, or CD19 +), and tumor cells expressing only CD20 (CD19 − / −, or CD19 −) were used. Used for analysis. In the presence of anti-CD20 rituximab, binding of CD16 by the Fc portion of the monoclonal antibody activates ADCC of hnCD16 expressing T cells, resulting in CAR-hnCD16 iT cells being both CD19 +/+ and CD19 − / − Raji cells. It is cytotoxic to (Fig. 25A). After 36 hours in the presence and absence of the anti-CD20 monoclonal antibody rituximab, the survival of target cells was quantified by Incucite Zoom ™. As shown in FIG. 25, CAR-hnCD16 iT cells mediated substantial CAR-mediated killing of the CD19 +/+ target and CD19-/-CD20 + target hnCD16 ADCC-mediated killing.

In further demonstration, peripheral blood-derived T cells were reprogrammed by targeted insertion of CD19 CAR into the T cell receptor A (TRAC) locus under transcriptional control of endogenous regulatory elements, and single-cell-derived clone TRAC. A targeted CAR expression master hiPSC strain (TRAC-CAR TiPSC) was generated. The clone was characterized by being pluripotent (> 95% SSEA4 / TRA181) and consisted of double allelic disruption at the TRAC locus. Despite the loss of TCR expression, during stage-specific differentiation, the TRAC-CAR hiPSC strain subsequently grows into CD34-positive cells differentiated into CD8-positive cells (95 ± 5%) with uniform CAR expression. Can be done. Approximately 28 days after T cell differentiation, induced cells were able to further proliferate and proliferate in suspension without TCR expression (FIG. 26). This synthetic T cell (TRAC-CAR iT) produces and proliferates effector cytokines (IFNγ, TNFα, IL2), degranulation (CD107a / b, perforin, granzyme B) (> 85% enters the cell cycle), and Upregulation of the activation markers CD69 and CD25 demonstrated in vitro function to induce an efficient cytotoxic T lymphocyte response to the CD19 antigen challenge. The production of IFNγ and TNFα by mature TRAC-CAR iT cells is significantly higher than that of primary T cells expressing CAR (Fig. 27). TRAC-CAR iT also targets tumors antigen-specifically and does not have the antigen-specific cytotoxic variation seen in primary T cells expressing CAR (FIG. 28). The transwell migration assay then evaluated the chemotaxis of D20 and D28 TRAC-CAR-iT cells in response to the thymus-derived chemokines shown in FIG. Growing T cells lose their ability to migrate to thymus-derived chemokines during maturation and are important for the ability of induced NK cells to promote migration, infiltration, and activation of T cells as set forth in the examples herein. Emphasize the meaning.

The TRAC-CAR TiPSC is then further engineered to express the hnCD16 receptor, and the resulting iPSC strain differentiates into synthetic T cells (TRAC-CAR-hnCD16 iT) lacking T cell signature TCR expression and is endogenous TCR. It expresses promoter-driven CAR and also expresses high-affinity non-cleavable CD16. In FIG. 35, the proliferative responses of TRAC-CAR iT cells and primary CAR-T cells to HLA-matched PBMC-derived T cells were compared using a mixed lymphocyte reaction assay. Response cells, namely TRAC-CAR iT cells and primary CAR-T cells, were each labeled with cell trace dyes and for dye dilutions that reflected cell proliferation due to alloreactivation to HLA-mismatched PBMC-derived T cells, 4 Evaluated by flow cytometry a day later. FIG. 35 shows that TRAC-CAR iT cells are not alloreactive to healthy HLA-mismatched cells, as opposed to primary CAR T cells that show pigment dilution after alloreactive triggers or associated cell proliferation. Is shown. Further analysis and characterization of the dual cytotoxic targeting of TRAC-CAR-hnCD16 iT and the ability of CAR to mitigate antigen escape in targeted tumor cells was performed and engineered as shown in FIG. TRAC-CAR-hnCD16 iT cells provide a secondary approach to target tumors by expressing hnCD16, which allows non-native ADCC in T cells.

Inducible T cells expressing CAR and hnCD16 not only target malignancies recognized by CAR, but such inducible T cells are native mature effector T cells (peripheral blood, cord blood, or other). It further acquires ADCC mechanisms that are not normally found in (primary T cells from tissues). In addition, iPSC-derived T cells have longer telomeres and less wasting compared to native mature effector T cells isolated from the primary source. Therefore, induced T cells obtained from iPSC differentiation expressing both CAR and hnCD16 act synergistically with therapeutic antibodies to address CAR-T cell antigen escaping through the acquired ADCC mechanism, thereby CAR-T. The removal of directional tumors can be enhanced. Exemplary therapeutic antibodies targeting liquid or solid tumors used in hnCD16-mediated ADCC include anti-CD20 (rituximab, beltuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), anti-Her2 (trastuzumab), anti-CD52. (Alemtuzumab), Anti-EGFR (Cellutuximab), Anti-CD38 (Daratumumab), Anti-SLAMF7 (Elotuzumab), and their humanized and Fc modified variants and functional equivalents, but not limited to these. In addition, bispecificity and triplet fusion of the Fab region of an antibody that targets tumor cell antigens such as anti-CD19, anti-CD20, and anti-CD33 antigens in combination with another Fab region that recognizes CD16 on NK cells. The design of specific antibodies results in stimulation of NK cells followed by killing of tumor cells.

Example 7-Induced NK cells for combination therapy with checkpoint inhibitor antagonists Checkpoints are cell molecules, often cell surface molecules, that can suppress or down-regulate the immune response when uninhibited. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene products, or reduce the activity of checkpoint molecules. The development of checkpoint inhibitors (CIs) that target PD1 / PDL1 or CTLA4 has changed the outlook for oncology, and these agents have resulted in long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint block therapy, and recurrence remains a major concern. Therefore, there is a need for new therapeutic approaches that have the ability to overcome CI resistance.

In this disclosure, the induced NK cells provided herein are shown to have both the ability to recruit T cells into the tumor microenvironment and the ability to enhance T cell activation at the tumor site. .. As shown in FIG. 17, activated iNK cells produce soluble factors that enhance T cell activation. In this assay, hnCD16 iNK cells were combined with K562 or K562 expressing high levels of PDL1 in the presence of ADCC-induced anti-PDL1 antibody. After overnight incubation, supernatants were collected and incubated on allogeneic donor T cells for 4 or 24 hours before flow cytometric staining of donor T cell expression of the T cell activation marker CD69. Induced NK cells generated through in vitro directed differentiation from induced pluripotent stem cells are negative for cell surface PD-1. Therefore, expression of PDL1 in induced NK cells did not have a discernible effect on the cytotoxicity of NK cells. Furthermore, the addition of anti-PDL1 antibody did not affect the cytotoxicity or degranulation of induced NK cells, suggesting that these cells are resistant to PDL1-PD1-mediated inhibition. In addition, activation of induced NK cells induces the secretion of soluble factors, including increased upregulation of CD69, as compared to primary NK cells, including peripheral blood (PB) or cord blood (UCB) NK cells. Demonstrated enhanced function of activating cells.

Using conventional transwell migration assays, the secretion of CCL3, CCL4, CXCL10, and other soluble factors by activation-induced NK cells of the invention promoted induced migration of activated T cells. In this assay, hnCD16 iNK cells were combined with SKOV-3 or SKOV-3-PDL1 expressing high levels of PDL1 in the presence of ADCC-induced anti-PDL1 antibody. After overnight incubation, supernatants were collected and incubated in the lower chamber of a standard transwell chemotaxis chamber for 24 hours with allogeneic donor T cells in the upper chamber. After incubation, migration of T cells to the lower chamber was quantified by flow cytometry. Therefore, as shown in FIG. 18, activated iNK cells (middle row) enhance T cell migration.

In addition, when activated, the induced NK cells herein are direct anti-antipathies demonstrated by cellular production of abundant inflammatory cytokines and chemokines, including interferon gamma (IFNγ), CCL3, CCL4, CXCL10, and CCL22. Shows tumor capacity. IFNγ plays an important role in the regulation of antitumor T cell activity. In this in vivo assay, 1E7 iNK cells I.I. P. (Intraperitoneal) injection or 5E6 activated T cell R. O (posterior orbital) injections, or both, were injected. After 4 days, peripheral blood and abdominal cavity were evaluated for the presence of T cells by flow cytometry. Compared to mice that received T cells but not induced NK cells, iPSC-derived NK cells I. P. In the mice that received the strain, the frequency of T cells in the peripheral blood decreased (Fig. 19A) and the frequency of T cells in the abdominal cavity increased (Fig. 19B). In FIGS. 19A and 19B, each data point represents an individual mouse. It was observed that induced NK cells promote T cell migration by recruiting activated T cells from the circulatory system to the peritoneum, as shown in this in vivo recruitment model.

In addition, utilizing an in vitro three-dimensional tumor spheroid model (further described in the Examples below), enhanced infiltration of T cells into tumor spheroids was observed in the presence of the provided induced NK cells. For 30,000 green fluorescently labeled T cells, alone with SKOV-3 microspheres (red core, lower panel in FIG. 20) or with 15,000 iNK cells (upper panel in FIG. 20). The combination was incubated and imaged for 15 hours. As shown in FIG. 20, T cells alone could not invade the center of spheroids, but the addition of iNK cells promoted T cell infiltration into tumor spheroids and tumor spheroid destruction.

The enhanced T cell infiltration and enhanced cytotoxicity of tumor spheroids when co-cultured with induced NK cells is shown in FIG. 23 by measuring the total integrated green fluorescence intensity within the largest red object mask. It is shown more quantitatively. Infiltration of T cells into SKOV-3 spheroids with induced NK cells (1: 1 ratio), CD3 + T cells (2: 1 ratio), or iNK (1: 1 ratio) + T cells (2: 1 ratio) 24 Measured in co-culture for hours, induced NK cells enhance T cell infiltration of tumor spheroids, as shown in FIG. 23A.

Co-culturing T cells and iPSC-derived NK cells in a 3D tumor spheroid model kills the tumor cells and promotes the production of IFNγ and TNFα. 7 days after incubation of effector cells with SKOV-3 spheroids (induced NK cells (1: 1 ratio), CD3 + T cells (2: 1 ratio), or iNK (1: 1 ratio) + T cells (2: 1 ratio)) , The supernatant was collected and evaluated for TNFα and IFNγ production (FIGS. 24A and B). Co-culture of T cells and iPSC-NK increases cytokine production in both CD4 ⁺ T cells and CD8 ⁺ T cells, and iPSC-induced NK cells synergize with T cells to kill solid tumors in a spheroid model. To enhance the production of IFNγ and TNFα.

Thus, by facilitating the recruitment of T cells to the tumor site and by enhancing the activation and infiltration of T cells, these functionally potent inducible NK cells contain checkpoint inhibitors and others. It has been shown to be capable of synergizing with T cell-targeted immunotherapy to alleviate local immunosuppression and reduce tumor mass. Taken together, these data support allogeneic combination therapies, including induced NK cells provided herein, and master as a renewable source for the production of induced NK cells in vitro. Provides evidence to support pluripotent cell lines. As shown herein, master pluripotent cell lines are used to obtain functionally enhanced inducible cell products for the purpose of allogeneic combination therapy, including one or more T cell targeting therapeutic agents. Can be accompanied by desirable genome editing.

Checkpoint inhibitors suitable for combination therapy with induced NK cells provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM). And Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctra4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl) ), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB , OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitors of inhibitory KIR (eg, 2DL1, 2DL2, 2DL3, 3DL1, 3DL2). , Not limited to these. Some embodiments of the combined therapy with the provided induced NK cells include an inhibitor that targets one checkpoint molecule. Some other embodiments of combination therapy with the provided induced NK cells include two, three, or more so that two, three, or more checkpoint molecules are targeted. Includes inhibitors of. In some embodiments, administration of two, three, or more checkpoint inhibitors in combination therapy with the provided induced NK cells is simultaneous or sequential. In some embodiments, the antagonist that inhibits any of the checkpoint molecules described above is an antibody. In some embodiments, the checkpoint inhibitory antibody is a mouse antibody, human antibody, humanized antibody, camel Ig, shark heavy chain only antibody (VNAR), Ig NAR, chimeric antibody, recombinant antibody, or antibody fragment thereof. Can be. Non-limiting examples of antibody fragments include Fab, Fab', F (ab) '2, F (ab) '3, Fv, single-stranded antigen-binding fragment (scFv), (scFv) 2, disulfide stabilization. Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and others that maintain binding specificity of the entire antibody Antibody fragments are included, they can be more cost effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one or two or three or more checkpoint inhibitors are atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monarizumab, nivolumab, pembrolizumab, and theirs. Includes at least one of the derivatives or functional equivalents.

Combination therapy with induced NK and one or more check inhibitors includes cutaneous T-cell lymphoma, non-hodgkin lymphoma, mycobacterial sarcoma, Paget's reticular disease, Cesarly syndrome, granulomatous flaccid skin, lymphoma-like scaly, chronic Mossy porridge, acute psoriasis lichen, CD30 + cutaneous T-cell lymphoma, secondary skin CD30 + large cell lymphoma, non-bacterial sarcoma CD30 skin large T-cell lymphoma, polymorphic T-cell lymphoma, Renato lymphoma, subcutaneous T-cell lymphoma, vascular central lymphoma, blast NK-cell lymphoma, B-cell lymphoma, Hodgkin lymphoma (HL), head and neck tumor, squamous epithelial cancer, horizontal print myoma, Lewis lung cancer (LLC), non- Small cell lung cancer, esophageal squamous epithelial cancer, esophageal adenocarcinoma, renal cell cancer (RCC), colon cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine cancer (SCNC), liver cancer Liquid cancers and solids, including, but not limited to, lymphoma, liver cancer, oral squamous epithelial cancer, pancreatic cancer, papillary thyroid cancer, intrahepatic bile duct cell cancer, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal cancer. It can be applied to the treatment of cancer.

When assessing responsiveness to combination therapies containing proven induced NK cells and anti-immunity checkpoint inhibitors, the responses were clinical benefit, survival to death, complete pathological response, pathological response. Quantitative measurement, clinical complete remission, clinical partial remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulatory tumor cell depletion, circulatory marker response, and RECIST (response endpoint in solid tumors) It can be measured by at least one of the criteria.

Example 3-Three-dimensional tumor spheroid model that mimics solid tumors for assessing in vivo cell infiltration and related functions An automated color-tagged three-dimensional tumor spheroid model (also called an in vivo tumorigenesis model) is available in-house Developed and utilized by IncuCyte ™ S3, it enables monitoring and quantification of cancer growth and morphological changes induced by the induced effector cells provided herein. This 3D tumor spheroid model can derive many in vitro solid tumor systems that are physiologically appropriate for assessing the in vitro function of effector cells, their interaction with tumor cells, and their tumor cell response. , Cannot be achieved with the 2D alternatives currently used in cancer immunology research.

Here, several solid tumor cell lines were first tested for their ability to form 3D tumor spheroids. Once an in vivo tumorigenesis model of the tumor is established, the effects of induced NK and / or T cells with or without enhancement in combination therapy with checkpoint inhibitors are analyzed and compared to target tumor cells. The in vivo ability of cells in killing, recruiting T cells (or other bystander effector cells), and tumor infiltration was evaluated.

Tumor spheroids are initiated in round-bottomed 96-well tissue culture plates in the presence of Matrigel using adherent cell lines previously transduced with the nuclear localization fluorescent protein NucLight ™ Red (NLR). Cell lines used to establish tumor spheroids include SKOV-3 (ovarian cancer), A549 (lung cancer), MCF7 (breast cancer), PANC1 (pancreatic cancer), and many others (eg, Arya et. Al., J. Chem. Pharma. Res., 2011, 3 (6): 514-520). Tumor spheroid formation progresses in about 3-4 days and is quantified by red fluorescence image analysis on the Incubite Zoom ™ imaging system. As an example, FIG. 20 shows SKOV3-NLR transduced cells forming spheres in the presence of 2.5% MTG in the presence of 2.5% MTG, with each frame at a specified time point by live kinetic imaging (top panel). Defined by the captured and applied algorithm mask (bottom panel).

For tumor cytotoxicity experiments, effector cells such as donated induced NK cells or T cells were added to a defined number of spheroids. After adding effector cells, disruption of tumor spheroid structure was visually monitored by Incubite ™ imaging and quantified by measurement of red fluorescence region, intensity, or integrated intensity. Infiltration of effector cell tumors can be quantified with commercially available image analysis software after exporting images from each point in time.

FIG. 21A shows the infiltration of induced NK cells into SKOV3-NLR spheroids over 48 hours. Induced NK cells are pre-labeled with RapidCytoLight ™ Green reagent and a mask (magenta) defines the spheroid core. Changes in spheroid size and integrated fluorescence intensity were continuously monitored over time (Fig. 21A).

Observation of T cell infiltration into spheroids was performed using T cells fluorescently labeled with a green fluorescent dye. Tumor infiltration by T cells is shown in FIG. 21B. A total of 30,000 green fluorescently labeled T cells were incubated with SKOV-3 microspheres (red nuclei) alone or in combination with 15,000 iNK cells (unlabeled) and imaged for 15 hours. .. T cell infiltration was determined by visualizing the accumulation of green T cells within the red tumor spheroids. As shown in FIG. 21B, T cells alone could not penetrate the center of the tumor spheroid, but the addition of iNK cells promotes T cell infiltration and tumor spheroid destruction, and in this application targeting solid tumors in vivo. Induced NK cells and checkpoint inhibitors were used to support combination therapy.

Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as a limitation to the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications to the disclosure disclosed herein can be made without departing from the scope and gist of the invention.

All patents and publications referred to herein indicate the level of skill in the art to which this disclosure relates. All patents and publications are incorporated herein by reference as if the individual publications were specifically and individually indicated as incorporated by reference.

The disclosures exemplified herein can be carried out without any one or more elements, limitations or limitations not specifically disclosed herein. Thus, for example, in each of the examples herein, the terms "contains," "consisting essentially of," and "consisting of" can all be replaced by any of the other two terms. The terms and expressions used are used as descriptive terms, not limiting, and the use of such terms and expressions excludes any equivalent of the features shown and described or any part thereof. Although not intended, it is recognized that various modifications are possible within the scope of the claimed disclosure. Thus, although the present disclosure is specifically disclosed by preferred embodiments and features as required, modifications and variations of the concepts disclosed herein can often be conceived by those skilled in the art, such. Modifications and modifications are within the scope of the invention as defined by the appended claims.

Claims (27)

A cell or a population thereof
(I) The cell is (a) an induced pluripotent cell (iPSC), a cloned iPSC, or an iPS cell line cell, or (b) an induced cell obtained by differentiating the cell of (a).
(Ii) The cell
(1) High affinity non-cleavable CD16 (hnCD16) or a variant thereof,
(2) A cell or population thereof comprising a chimeric antigen receptor (CAR) and one or both of an exogenous cytokine expressed on the cell surface or a partial or complete peptide of the receptor. 1. The inducing cells of (i) and (b) are hematopoietic cells and contain longer telomeres as compared to their native corresponding cells obtained from peripheral blood, cord blood, or any other donor tissue. A cell or a population thereof according to. The cells
(I) B2M null or low,
(Ii) CIITA null or low,
(Iii) Introduced expression of HLA-G or non-cleavable HLA-G,
(Iv) At least one of the genotypes listed in Table 1,
(V) Deletion or underexpression in at least one of the genes in the TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and chromosome 6p21 regions.
(Vi) HLA-E, 41BBL , CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptors, engagers, and bispecific or The cell according to claim 1 or a cell thereof, further comprising one or more of introduced or increased expression in at least one of the surface-triggered receptors for binding to a multispecific or universal engager. Group. The cells are derived NK or induced T cells and are compared to their native counterpart cells obtained from peripheral blood, cord blood, or any other donor tissue.
(I) Improved persistence and / or survival,
(Ii) Increased resistance to innate immune cells,
(Iii) Increased cytotoxicity,
(Iv) Improved tumor penetration,
(V) Enhanced or acquired ADCC,
(Vi) Enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site,
(Vii) Enhanced ability to reduce tumor immunosuppression, as well as (vii) Improved ability to rescue tumor antigen escapes,
The cell or population thereof according to claim 1 or 3, which has at least one of the characteristics including. The high affinity non-cleavable CD16 (hnCD16) or a variant thereof
(A) F176V and S197P of the external domain domain of CD16,
(B) Complete or partial external domain derived from CD64,
(C) Non-natural (or non-CD16) transmembrane domain,
(D) Non-natural (or non-CD16) intracellular domain,
(E) Non-natural (or non-CD16) signaling domains,
Claim 1 comprises (f) a non-natural stimulus domain and (g) at least one of a transmembrane, signal transduction, and stimulus domain that is not derived from CD16 but is derived from the same or different polypeptide. A cell or population thereof according to. (A) The non-natural transmembrane domain is CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, Derived from CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide Or,
(B) Is the non-naturally stimulating domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide? ,
(C) Whether the unnatural signaling domain is derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. Or (d) the cell of claim 5, wherein the unnatural transmembrane domain is derived from NKG2D, the unnatural stimulating domain is derived from 2B4, and the unnatural signaling domain is derived from CD3ζ. Or its group. The CAR
(I) T cell-specific or NK cell-specific,
(Ii) Bispecific antigen-binding CAR,
(Iii) Switchable CAR,
(Iv) Dimerized CAR,
(V) Split CAR,
(Vi) Multiple chain CAR,
(Vii) Inducible CAR,
(Viii) Co-expressed with another CAR,
(IX) Co-expressed with a partial or complete peptide of a cell surface-expressed exogenous cytokine or receptor thereof, optionally within a separate construct or within a bicistronic construct,
(Xi) Co-expressed with a checkpoint inhibitor, as needed, in a separate construct or in a bicistronic construct,
(Xii) CD19 or BCMA specific and / or (xiii) ADGRE2, carbonate dehydration enzyme IX (CALX), CCR1, CCR4, cancer fetal antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, cytomegalovirus (CMV) -infected cell antigen, epithelium Glycoprotein 2 (EGP2), Epithelial Glycoprotein-40 (EGP-40), Epithelial Cell Adhesion Molecules (EpCAM), EGFRvIII, Receptor Tyrosine Protein Kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB Folic Acid Binding Protein (FBP), fetal acetylcholine receptor (AChR), folic acid receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcription enzyme (HTERT), ICAM-1, Integrin B7, Interleukin-13 Receptor Subunit Alpha-2 (IL-13Rα2), κ-Light Chain, Kinase Insertion Domain Receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, Mutin 1 (Muc-1), Mutin 16 (Muc-16), Mesocerin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testile antigen NY-ESO-1, cancer fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), Specific to any of tumor-related glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigen What is
The CAR of any one of (i) to (xiii) is optionally inserted into the TRAC locus and / or driven by the endogenous promoter of TCR and / or said TCR. The cell or population thereof according to claim 1, wherein is knocked out by the CAR insertion. The cell further comprises a partial or complete peptide of an exogenous cytokine or receptor thereof expressed on the cell surface, the exogenous cytokine or receptor thereof.
(A) Contains at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (b).
(I) Co-expression of IL15 and IL15Rα by suing a self-cleaving peptide,
(Ii) A fusion protein of IL15 and IL15Rα,
(Iii) An IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been cleaved,
(Iv) A fusion protein of the membrane-bound Shishi domain of IL15 and IL15Rα,
(V) A fusion protein of IL15 and IL15Rβ,
A fusion protein of (vi) IL15 and the common receptor γC, wherein the common receptor γC is a natural or modified fusion protein, and at least one of the homodimers of (vi) IL15Rβ. Including, any one of (i)-(vii) can be co-expressed with CAR within a separate construct or within a bicistronic construct.
If necessary,
(C) The cell or population thereof that is transiently expressed, according to claim 1. The cells are induced NK cells or induced T cells.
(I) The induced NK cells can mobilize T cells and / or migrate to the tumor site.
(Ii) The cell or population thereof according to claim 3, wherein the induced NK or induced T cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. The checkpoint inhibitors are PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73. , CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoin) The cell or population thereof according to claim 7 or 9, which is an antagonist to one or more checkpoint molecules comprising acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. The checkpoint inhibitor
(A) Atezolizumab, Abelumab, Durvalumab, Ipilimumab, IPH4102, IPH43, IPH33, Lilimumab, Monarizumab, Nivolumab, Pembrolizumab, and one or more of their derivatives or functional equivalents, or (b) Atezolizumab, Nivolumab, The cell or population thereof according to claim 9, which comprises at least one of pembrolizumab. The inducible cells are inducible CD34 cells, inducible hematopoietic stem cells and progenitor cells, inducible hematopoietic pluripotent progenitor cells, inducible T cell progenitor cells, inducible NK cell progenitor cells, inducible T cells, inducible NKT cells, inducible NK cells, or inducing. The cell or population thereof according to claim 2, which comprises B cells. The cells
1) Claim 1 comprising (i) one or more exogenous polynucleotides integrated into one safe harbor locus, or (ii) more than two exogenous polynucleotides integrated into different safe harbor loci. A cell or a population thereof according to. 13. The cell or population thereof according to claim 13, wherein the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. The cell or population thereof according to claim 15, wherein the safe harbor locus TCR is a constant region of TCR alpha. A composition comprising the cell or a population thereof according to any one of claims 1 to 15. A composition for therapeutic use comprising the inducer cell according to any one of claims 1 to 15 and one or more therapeutic agents. One or more of the therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors. 17. The composition of claim 17, comprising a vector, antibody, chemotherapeutic agent or radioactive moiety, or immunomodulatory agent (IMiD) containing the polypeptide of interest. The checkpoint inhibitor
(A) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96 , CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha) ), TLR3, VISTA, NKG2A / HLA-E, or one or more antagonist checkpoint molecules, including inhibitory KIR.
(B) Atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, nivolumab, monarizumab, nivolumab, pembrolizumab, and one or more of their derivatives or functional equivalents.
(C) The composition of claim 18, comprising atezolizumab, nivolumab, and at least one of pembrolizumab. The antibody
(A) Anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibody,
(B) Rituximab, Bertzzumab, Ofatumumab, Ubrituximab, Okaratsuzumab, Obinutuzumab, Trastuzumab, Pertuzumab, Alemtuzumab, Serutuximab, Zinutuximab, Abelumab, Daratumumab, Abelumab, Daratumumab, Isatuzumab, Daratumumab The composition of claim 18, comprising variants or fragments, and one or more of their functional equivalents and biosimilars, or (c) daratumumab. Therapeutic use of the composition by introducing the therapeutic composition according to any one of claims 16 to 20 into a subject suitable for adoptive cell therapy, wherein the subject is an autoimmune disease, blood. Have a malignant tumor, solid tumor, cancer, or viral infection, therapeutic use. The method for producing an induced cell according to any one of claims 1 to 15, wherein the iPSC comprises differentiating the iPSC.
(I) High affinity non-cleavable CD16 (hnCD16) or a variant thereof,
(Ii) Includes a chimeric antigen receptor (CAR) and one or both of a cell surface expressed exogenous cytokine or a partial or complete peptide of that receptor, as required.
(1) B2M null or low,
(2) CIITA null or low,
(3) Introduced expression of HLA-G or non-cleavable HLA-G,
(4) At least one of the genotypes listed in Table 1,
(5) Deletion or expression reduction in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.
(6) HLA-E, 41BBL , CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptors, engagers, and bispecific or A method comprising one or more of introduced or increased expression in at least one of the surface-triggered receptors for binding to a multispecific or universal engager. Genome manipulation of cloned iPSC
(I) High affinity non-cleavable CD16 (hnCD16) or a variant thereof,
(Ii) Further comprising introducing expression with one or both of the chimeric antigen receptor (CAR) and an exogenous cytokine of cell surface expression or a partial or complete peptide of that receptor, as required. hand,
(1) Knock out B2M null or
Includes (2) knocking out CIITA or (3) introducing expression of HLA-G or non-cleavable HLA-G.
22. The CAR and the partial or complete peptide of the exogenous cytokine or receptor thereof expressed on the cell surface are co-expressed within a separate construct or within a bicistronic construct. A method of producing an inducible cell. 22. The method of producing an induced cell according to claim 22, wherein the genome manipulation comprises targeted editing. The targeted edit comprises deletion, insertion, or indel, and the targeted edit is a CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods. 24. The method of producing an indel cell according to claim 24. Editing of a clone iPSC mediated by CRISPR, wherein the edited clone iPSC
(A) (i) High affinity non-cleavable CD16 (hnCD16) or a variant thereof,
(Ii) Chimeric antigen receptor (CAR) and, optionally, cell surface-expressed exogenous cytokines or partial or complete peptides of those receptors, or
(B) Containing at least one of the genotypes listed in Table 1.
A CRISPR-mediated clone in which the CAR has been inserted at the TRAC locus as needed and / or driven by the endogenous promoter of the TCR and / or the TCR has been knocked out by the CAR insertion. Editing of iPSC. A method of escaping tumor antigens and / or preventing or reducing tumor recurrence in a subject under treatment.
(A) (i) High affinity non-cleavable CD16 (hnCD16) or a variant thereof,
(Ii) Chimeric antigen receptor (CAR) and, optionally, cell surface-expressed exogenous cytokines or partial or complete peptides of those receptors, or
(B) Effector cells containing at least one of the genotypes listed in Table 1 and
An antigen targeted by an antibody comprising administering an antigen-specific monoclonal antibody, or a humanized or Fc-modified variant or fragment thereof, any of their functional equivalents and biosimilars, is the CAR. A method different from the tumor antigens recognized by.